Optimal Conditions For Chromatographic Separation Research Articles

RP-UFLC based Bioanalytical Method Development, Optimization, and Validation for the Estimation of Isradipine in Rabbit Serum

Introduction: The objective of this study is to provide a rapid, sensitive, consistent, and costeffective method for quantifying isradipine using ultra-fast liquid chromatography. Methods: Quality by Design principles will form the basis of this approach, grounded on response surface analysis. Shimadzu liquid chromatographic system equipped with a photodiode array detector and LC solution software was used to conduct the RP-UFLC method development and validation. An ODS C18 (250 x 4.6 mm; 5 μm) UFLC column was used to complete the analysis. The RSM methodology utilized a central composite design to perform the optimization studies. Results: The mobile phase ratio and flow rate were considered crucial method parameters, as well as the peak area, retention time, and USP plate were considered critical analytical attributes. The optimal conditions for chromatographic separation were followed using 80% acetonitrile and water (20% v/v) as mobile phase, a 1 mL/min flow rate, an injection volume of 20 μL, 40°C of column oven temperature, and maximum absorption at λmax 254 nm using graphical optimization technique. When examining concentrations between 5 and 150 ng/mL, linearity was observed with an R2 of 0.999. The method created was validated by employing stability testing per the recommendations provided by ICH Q2 (R1). The analysis of blood serum was modified so that it could be used to examine the pharmacokinetic parameters. Conclusion: As a result, high accuracy, precision, sensitivity, linearity, and robustness were established for predicting the amount of isradipine present in its freeze-dried nano-formulations.

Method Optimisation in Hydrophilic-Interaction Liquid Chromatography by Design of Experiments Combined with Quantitative Structure–Retention Relationships

Accurate prediction of the separation conditions for a set of target analytes with no retention data available is fundamental for routine analytical assays but remains a very challenging task. In this paper, a quality by design (QbD) optimisation workflow capable of discovering the optimal chromatographic conditions for separation of new compounds in hydrophilic-interaction liquid chromatography (HILIC) is introduced. This workflow features the application of quantitative structure−retention relationship (QSRR) methodology in conjunction with design of experiments (DoE) principles and was used to carry out a two-level full factorial DoE optimisation for a mixture of pharmaceutical analytes on zwitterionic, amide, amine, and bare silica HILIC stationary phases, with mobile phases containing varying acetonitrile content, mobile phase pH, and salt concentration. A dual-filtering approach that considers both retention time (tR) and structural similarity was used to identify the optimal set of analytes to train the QSRR in order to maximise prediction accuracy. Highly predictive retention models (average R2 of 0.98) were obtained and statistical analysis of the prediction performance of the QSRR models demonstrated their ability to predict the retention times of new compounds based solely on their molecular structures, with root-mean-square errors of prediction in the range 7.6–11.0 %. Further, the obtained retention data for pharmaceutical test compounds were used to compute their separation selectivity, which was used as input into a DoE optimiser in order to select the optimal separation conditions. Experimental separations performed under the chosen optimal working conditions showed good agreement with the theoretical predictions. To the best of our knowledge, this is the first study of a QbD optimisation workflow assisted with dual-filtering-based retention modelling to facilitate the method development process in HILIC.

Development and Validation of four Nitrosamine Impurities Determination Method in Medicines of Valsartan, Losartan, and Irbesartan with HPLC-MS/MS (APCI).

Since 2018, regulation and control of genotoxic nitrosamine impurities levels have been a mandatory quality and safety characteristic for various drugs. The main issue of nitrosamine determination in drugs is a low sensitivity of the existing methods and a continuously extending list of controlled compounds. The reason is the low safe daily dose of these impurities and chromophores’ absence within their structure. Development and validation of a method for nitrosamine impurities (regulated by the regulatory authorities) determination in Valsartan, Losartan, and Irbesartan using high-performance liquid chromatography with mass spectrometry detection. An Agilent Infinity II chromatographic system with a mass spectrometric detector (MSD 6460 Triple Quad) and atmospheric pressure chemical ionization was used in this study. During the development of a method, the optimal conditions for chromatographic separation (composition of mobile phases, gradient parameters) were selected, as well as the parameters of mass spectrometric detection were optimized. The usage of chemical ionization made it possible to achieve the method’s maximum sensitivity concerning the studied nitrosamines, and the optimized parameters of mass spectrometric detection made it possible to get rid of the matrix effect. The absence of additional stages of purification and concentration can significantly reduce the total time of the analysis, which is a significant advantage of nitrosamine’s advanced determination method. The resulting method was validated for specificity, linearity, LOQ, LOD, accuracy, and precision. Resulting method met all acceptance criteria and can be used for routine quality control of Valsartan, Losartan, and Irbesartan pharmaceutical substances.

Development and Validation of an RP-HPLC Method for Determination of Atorvastatin and its Hydroxyl Metabolites in Human Plasma

Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.

Экспресс-идентификация и определение натамицина в пищевых продуктах методом ультравысокоэффективной жидкостной хроматографии/квадруполь-времяпролетной масс-спектрометрии высокого разрешения

Предложена методика экспресс-идентификации и определения полиенового антибиотика натамицина в пищевых продуктах методом ультравысокоэффективной жидкостной хроматографии в сочетании с квадруполь-времяпролетной масс-спектрометрией высокого разрешения. Диапазон определяемых содержаний натамицина составил от 0.5 до 100 нг/г. Для определения обнаруженного натамицина предложено использовать метод стандартной добавки. Относительное стандартное отклонение результатов анализа не превышало 0.12. Продолжительность идентификации проб 20 мин, определения натамицина – 40 мин

Development of method of quantitative determination of cardiazol substance with using highly efficient liquid chromatography

The aim. Development of methods of quantitative determination of Cardiazol substance using high-performance liquid chromatography.Materials and methods. The method of high-performance liquid chromato-graphy (HPLC) was to determine of quantitative determination of Cardiazol substance ([3-Allil-4-(41-methoxyphenyl)-3H-thiazole-2-ylidene]-(32-trifluoro-methylphenyl)amine hydrobromide) using Shimadzu Nexera X2 LC -30AD (Shimad-zu, Japan). The acetonitrile of the HPLC grade (Sigma-Aldrich GmbH, Switzerland) was used in the work and other chemicals and solvents were of analytical grade. The test substance was diluted in acetonitrile at a final concentration of 400 μg/ml.Results and discussion. The method of quantitative determination of Cardiazol substance with the help of highly effective liquid chromatography is developed. The developed conditions of testing are selected experimentally. The following optimal conditions for the chromatographic distribution were found: column C8 (250 *4.6 mm; speed of the mobile phase 1 ml/min; thermostat temperature of the column 35 ° C; detecting wavelength 300 nm; holding time of the test compound is 13.9 min. Suitability of determination methods.The following optimal conditions for chromatographic separation were revealed: a C8 column (250*4.6 mm, a mobile phase speed of 1 ml/min, a column thermostat temperature of 35 °C, a detection wavelength of 300 nm. Under the proposed conditions, the retention time of the tested component is 13.9 minutes. The performance of the column was determined for its main indicators, such as the theoretical number of plates (over 65000) and the coefficient of symmetry (about 1.00). The method of quantitative determination was tested in accordance with the recommendations of the Ukrainian and European Pharmacopoeia. The proposed method meets all requirements. The method has been tested for the effects of various factors such as flow rate, the composition of the mobile phase and the temperature of the column thermostat. It is established that the influence of these factors is insignificant and does not affect the results obtained by this method.Conclusions. An analytical method for quantitative determination of Сardiazole substance with cardioprotective action has been developed on the basis of the high-performance liquid chromatography method. The conditions for chromatographic analysis (HPLC) were standardized. Requirements for the test Checking the suitability of the chromatographic system were established. The statistical processing of the results of the experiment shows that the relative uncertainty of the average result was within the permissible limits. The developed method for the determination of Сardiazole will be used for further study of substance as a component of various dosage forms

DETERMINATION OF SODIUM GLUTAMATE BY THIN LAYER CHROMATOGRAPHY METHOD WITH FLUORESCENT DETECTION

The spectral and luminescent properties of Tb(III) complex with ciprofloxacin and monosodium glutamate (MSG) were studied. The stoichiometric composition of complex (Tb(III) : ciprofloxacin : MSG = 1:2:1) was determined by the Bent-French method. Its spectral and luminescent characteristics in the phase of sorbent (the maximum wavelength of the luminescence and luminescence excitation, the lifetime of luminescence) were defined. It was established that effective energy transfer takes place from the ligand excited state to lanthanide ion in Tb(III) – ciprofloxacin – MSG complex resulting in intensive sensitized luminescence. The optimal conditions of chromatographic separation of monosodium glutamate (stationary and mobile phase, the sample volume applied to the plate, the optimal concentration of a luminescent detection reagent (Tb(III), ciprofloxacin, MSG) were selected. The maximal luminescence intensity was occurred at 1·10–3 mol/L Tb (III) in neutral solutions at pН 6,5 – 7,5. The maximal luminescence intensity of Tb(III) – ciprofloxacin – MSG complex was observed at 2.10-3 mol/L ciprofloxacin and at 1·10–3 mol/L MSG. A thin layer chromatography method for determining of monosodium glutamate in dried squids with using Tb (III) – ciprofloxacin as a fluorescent detection reagent has been proposed. Sodium glutamate was found in all commercial samples of dried squid. This means that some manufacturers do not indicate the presence of this food additive on the label of the products in the ingredient list in any packaged food in contrast of food labeling law.

Determination of Quinine and its Derivatives with High-Performance Liquid Chromatography

This article presents the results deriving from research made on the development of optimal conditions for chromatographic separation and determination of such compounds as: quinine, quinidine, and acetylquinidine. The compounds subject to analysis are used in the treatment of malaria. Three stationary phases were used in the examination: octadecyl, which is perceived by analysts as the standard one, and two aryl phases (benzylpropyl and naphthylpropyl). For the mobile phases, the following compounds were adopted: methanol, acetonitrile, and a mixture of acetonitrile/water/formic acid. The best separation of quinine, quinidine, and acetylquinidine was observed in the naphthylpropyl column with the application of methanol as the mobile phase. The coefficients of separation of these compounds with the flow of methanol of 0.8 mL min−1 were 1.17 and 1.14, respectively. However, no proper separation of the compounds was observed in the octadecyl column, regardless of the mobile form applied.

Separation of Benzyl Derivatives of Germanium for HPLC

The paper presents interactions of mono-, di-, tri-, and tetrabenzyl derivatives of germanium with stationary phase surface and results from these processes of determination. As a final result of the work, optimal conditions of chromatographic separation and determination of these compounds by high-performance liquid chromatography (HPLC) method are proposed. During investigations, different types of stationary phase (including octadecyl, octyl, and chemically bonded aryl phases) and mobile phases are considered. As a result of the investigation, the highest selectivity is showed chemically by bonded aryl stationary phase combined with a mobile phase consisting of acetonitrile (capacity factors of di-, tri-, and tetrabenzyl derivatives of germanium k(1) = 0.86, k(2) = 2.53, k(3) = 6.45). However, octadecyl phase, which is considered as reference phase, exhibits the lowest separation factors of determined benzylgermanium compounds (k(1) = 9.12, k(2) = 37.62, k(3) = 70.43).

SEPARATION AND DETERMINATION OF ALKYLFURYLSILANE ISOMERS FOR HPLC

In this paper are presented optimal conditions of chromatographic separation of two isomers: γ-(chloropropyl)-3-furyldimethylsilane and [2-chloro(1-methyl)ethyl](3′-furyl)dimethylsilane. These isomers show the ability of modification of the surface of silica gel. In the investigation were tested three stationary phases and three mobile phases: dichloromethane, methanol, and a mixture of dichloromethane and hexane (80/20, v/v), in different flow intensities. An application of octadecyl stationary phase, which is recommended as a standard phase, did not yield separation of the isomers, independently of composition and flow of a mobile phase. Better results, but with asymmetric peaks have been obtained on the octyl phase. Finally, aryl chemically bonded phases permitted acquiring good separation of the isomers in a time shorter than 25 min. The best separation factor (1.11) has been obtained on the chemically bonded aryl phase using dichloromethane with flow of 0.1 mL · min−1. Therefore, the best selectivity in separation of above mentioned compounds exhibited the aryl chemically bonded phase.

Separation of Azepinio‐Methyl Derivatives of ES‐Silanates by the Use of Aryl Stationary Phases in HPLC

Optimal conditions of chromatographic separation and determination of chosen higher coordinated compounds belonging to a group of ES‐silanates have been elaborated. In the analysis were considered: 5λ‐Si‐[(azepinio)methyl]bis[2‐methyllactic(2−)‐01, 02]silicate and 5λ‐Si‐[(azepinio)methyl]bis[2‐methylglycolic(2‐)‐01, 02]silicate. Investigations were carried out applying three stationary phases (phenylbutyl, naphthylpropyl, and hypercarb), two mobile phases (acetonitrile, dichloromethane) and various flows. The shortest retention times were observed when a hypercarb column containing graphitized coal was used. However, the highest separation factor (α=3.05) was obtained using the naphthylpropyl column and the mobile phase consisting of pure acetonitrile (flow 1 mL · min−1).

Separation of Biologically Active Isomers of (E)‐N‐Meta‐ and Para‐Nitroazastilbenes by the HPLC Technique

Results of investigations concerning the chromatography of bromides of (E)‐N‐m‐ and p‐nitrobenzyl‐4′‐hydroxy‐3′‐methoxystilbazoles‐4 are presented. Analyzed compounds, similarly to other azastilbene halogenides show antimicrobial activity. Optimal conditions of chromatographic separation and determination of two mentioned isomers have been elaborated. In the investigation three stationary phases (octadecyl, octyl, and naphthylpropyl), two mobile phases (acetonitrile, dichloromethane) and various flow were considered. The best selectivity and the highest separation factor, α=1.74, were obtained using a naphthylpropyl column and 100% acetonitrile, as the mobile phase. An application of an octadecyl phase, recommended by numerous analysts as standard, allowed only the observation of an existence of two compounds, but did not yield satisfactory results.

Separation of Hexabenzylgermanium Derivatives using Aryl Stationary Phases for HPLC

This paper is a continuing investigation concerning optimization of the chromatography of hexabenzylgermanium derivatives. Optimal conditions of chromatographic separation and determination for hexabenzyldigermoxane and hexabenzyldigermanium have been elaborated, utilizing interactions of π‐π type between stationary phase and analyzed compound. Because of partial retention of these compounds on a chromatographic column, numerous problems with their determination can appear. During the investigation three aryl stationary phases and various mobile phases were taken into consideration. Obtained results showed, unanimously, that the best selectivity exhibited a column containing porous graphitizing coal. The highest separation factor, 1.73, has been obtained using this column and a mobile phase consisting of aqueous acetonitrile. A slightly lower separation factor has been obtained with the use of aryl stationary phase RP Si–NAF. All obtained results showed a dominating influence of interactions π‐π in the investigated chromatography process.

Adsorption semiconductor detector for malfunction diagnosis of high voltage transformers

The basis of the transformer diagnosis is a correlation between a particular malfunction and the content of certain gases dissolved in the transformer oil. A chromatography detector based on a semiconductor sensor was developed to provide for selective and sensitive analysis of H 2, CH 4, C 2H 6, C 2H 4, C 2H 2, CO—crucial gases for the transformer diagnosis. The semiconductor sensor was based on metal oxides. The sensor sensitivity was increased by doping the sensitive layer with Pt, Pd or their mixtures. The sensor impregnated with 0.84×10 −2 M PdCl 2 solution satisfied the requirement of simultaneous and sufficient sensitivity to all of the gases. The optimal conditions of chromatographic separation of the above gases were investigated, and the overall time of analysis was decreased by adding 10-port valves to the chromatography setup. The sensor stability was also investigated, and it was shown that a stable operation of the developed detector can be provided.

Determination of Food Dyes by Thin-Layer Chromatography with Cyclodextrin Mobile Phases

metrically by the decrease in the absorbance ofthe solution. In the flow-injection mode, we studiedthe effect of the length of the capillary on peakbroadening and found the optimal concentrations ofthe reagents that provide the maximum analyticalsignal. The interfering effect of some anions andcations was examined. Optimal conditions ofchromatographic separation were selected. The detec-tion limit of nitrite in water was one-fifth of the max-imum permissible concentration ( 1.5 × 10

Rapid and sensitive method for measurment of hyaluronic acid and isomeric chondroitin sulfates using high-performance liquid chromatography

A high-performance liquid chromatographic method for the separation and analysis of the unsaturated tetrasaccharide and hexasaccharide from Streptomyces hyaluronidase (S.HAase) enzyme digestion products of hyaluronic acid (HA) and standard unsaturated disaccharides 2-acetamido-2-deoxy-3-O-(β- D-gluco-4-enepyranosyluronic acid)- d-galactose (ΔDi-0S), 2-acetamido-2-deoxy-3-O-(β- d-gluco-4-enepyranosyluronic acid)-4-O-sulfo- d-galactose (ΔDi-4S) and 2-acetamido-2-deoxy-3-O-(β- d-gluco-4-enepyranosyluronic acid)-6-O-sulfo- d-galactose (ΔDi-6S) is described. An amino phase chemically bonded to silica with a particle diameter of 6 μm was used as the column. The composition and the pH of the mobile phase were systematically varied to determine the optimal chromatographic conditions for separation and analysis of the compounds. For HA, a complete separation was accomplished in less than 12 min with a practical detection limit of 100 ng. Separation of the disaccharides also required less than 15 min with detection limits of 10 ng for ΔDi-0Sand 25 ng each for ΔDi-4S and ΔDi-6S. This chromatographic method represents a significant improvement over existing methods. It allows the simultaneous separation and analysis of HA and chondroitin sulfate isomers (after digestion of the latter with chondroitinase) at a higher speed, and with more sensitivity and efficiency.