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1

High-performance liquid chromatography evaluation of lipophilicity and QSRR modeling of a series of dual DNA gyrase and topoisomerase IV inhibitors

Dobričić Vladimir, Savić Jelena, Tomašič Tihomir, Durcik Martina, Zidar Nace, Peterlin Mašič Lucija, Ilaš Janez, Kikelj Danijel, Čudina Olivera

Acta Chromatographica

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2024

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Vol. 36, no. 1

45--51

EN

Bacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and represent important antibacterial drug targets. To be successful as drug candidates, newly synthesized compounds must possess optimal lipophilicity, which enables efficient delivery to the site of action. In this study, retention behavior of twenty-three previously synthesized dual DNA gyrase and topoisomerase IV inhibitors was tested in RP-HPLC system, consisting of C8 column and acetonitrile/phosphate buffer (pH 5.5 and pH 7.4) mobile phase. logD was calculated at both pH values and the best correlation with logD was obtained for retention parameter ϕ0, indicating that this RP-HPLC system could be used as an alternative to the shake-flask determination of lipophilicity. Subsequent QSRR analysis revealed that intrinsic lipophilicity (logP) and molecular weight (bcutm13) have a positive, while solubility (bcutp3) has a negative influence on this retention parameter.

2

Method development and validation for simultaneous determination of Eletriptan hydrobromide and Itopride hydrochloride from fast dissolving buccal films by using RP-HPLC

Siddique Waqar, Sarfraz Rai Muhammad, Zaman Muhammad, Khan Riffat, Gul Maria, Asghar Farhan, Malik Tangina, Saif Asiya, Shamim Qurat-Ul-Ain, Salawi Ahmad, Alshamrani Meshal, Almoshari Yosif, Sabei Fahad Y.

Acta Chromatographica

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2023

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Vol. 35, no. 4

310--318

EN

One of the most effective, rapid, and simple methods reversed-phase high-performance liquid chromatography (RP-HPLC) was used for simultaneous development and validation of Eletriptan hydrobromide (ELE HBR) and Itopride hydrochloride (ITP HCL) in combination. The method was validated based on the regulations of United States Pharmacopeia (USP) and International Conference on Harmonization (ICH) guidelines. Separation of both drugs was achieved within approximately 5 min by using a mobile phase made up of a 70:30 ratio of phosphate buffer and acetonitrile having a flow rate of 1 mL min−1. Furthermore, a comprehensive study was conducted on precision, accuracy, linearity, inter-day, intra-day studies, an assay of formulated films, and stability studies of combined prepared film. Co-efficient of correlation ranged between 0.9993, and 0.9965 for ELE HBR and ITP HCL respectively. The accuracy of the developed method was accurate as drug recoveries in both cases of ITP HCL, and ELE HBR falls between (99.87, 99.96, and 99.84%) to (99.81, 99.12, and 98.44%) respectively having a concentration range of solutions between 10, 30 and 50 μg mL−1 dilution. Films developed by using both drugs in combination were then validated for assay studies, and it was found that substantial results of 99.05%, and 99.87% were found in the case of ITP HCL and ELE HBR respectively. The stability of the solution and mobile phase showed the method's accuracy as the results were 97% for ITP HCL and 99% for ELE HBR. The proposed method developed for simultaneous determination of ITP HCL and ELE HBR was developed and validation and no interaction of any excipient were found.

3

Development and validation of RP-HPLC method for quantification of trace levels of topical corticosteroids in ambiphilic cream

Ivković Branka, Crevar Milkica, Cvetanović Anka, Ubavkić Katarina, Marković Bojan

Acta Chromatographica

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2023

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Vol. 35, no. 1

46--51

EN

Corticosteroids are anti-inflammatory and immunosuppressant drugs. Topical corticosteroids formulations (ointments, creams, gels) are used in the treatment of different types of dermatitis and urticaria. Considering their therapeutic and whitening effects, they are frequently used for counterfeiting of cosmetic products. Corticosteroids can cause different local and systemic side effects. HPLC method is often chosen for their analysis, because it is selective, sensitive, precise, simple and fast. The aim of this study was optimization and validation of RP-HPLC method with UV detection for determination of trace levels of corticosteroids in ambiphilic creams. This method is used for qualitative and quantitative analysis of evaluated corticosteroids. Mometasone furoate, hydrocortisone acetate, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone dipropionate and triamcinolone acetonide were evaluated. Separation was performed on Inertsil® ODS-3V 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase was mixture of acetonitrile and water 50:50 (v/v) with gradient elution and flow rate 1 mL min⁻¹. Column temperature was held on 40 °C and UV detection was performed at 240 nm. Selectivity, linearity, accuracy, precision and limit of quantification (LOQ) were evaluated. Method is selective because ambiphilic cream base peaks and corticosteroids peaks were not overlapping. Linearity was confirmed since correlation coefficient was 1 for all compounds. Accuracy and precision were evaluated for hydrocortisone acetate and betamethasone dipropionate. Determined Recovery values were in range of 70–130%. Both RSD values (21.46% and 9.59%) were lower than 30%. Method is highly sensitive since LOQ concentrations were in ng mL⁻¹ range. All evaluated parameters of validation were in accordance with regulatory requirements. Validated RP-HPLC method can be used for qualitative and quantitative analysis of selected corticosteroids in ambiphilic creams.

4

Analytical method validation on simultaneous estimation of Ozenoxacin and Benzoic acid in pharmaceutical formulation

Ramireddy Amarnath Reddy, Behara Dilip Kumar

Acta Chromatographica

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2023

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Vol. 35, no. 3

278--285

EN

In this study, an accurate, simple, economical and precise Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) method was developed for the simultaneous estimation of Ozenoxacin and Benzoic Acid in a pharmaceutical cream formulation, according to the International Conference on Harmonisation (ICH) guidelines. Chromatographic separation was achieved by gradient elution, on RP-HPLC Instrument, equipped with column C8 (150 mm × 4.6 mm, 5 μm particle size) using Ultra Violet (UV) detector at 235 nm wavelength, by using Mobile Phase A: triethylamine, trifloroacetic acid and water (1:1:1000) and Mobile Phase B: methanol and Diluent: water, acetonitrile and triethylamine (500:500:1), at flow rate 0.8 mL min⁻¹; injection volume of 20 μL; column oven temperature 45 °C and sample temperature: 25 °C; Run time: 15 min. All the validation parameters were within the acceptance criteria, as per ICH requirements, for Ozenoxacin and Benzoic acid. Consequently, this method has found to be validated, simple, rapid and successfully applicable, to the simultaneous estimation of Ozenoxacin and Benzoic acid by RP-HPLC, for routine analytical testing in quality control, with a run time of 15 min and for future research studies. Forced degradation of Ozenoxacin cream 1% w/w formulation was performed and found that validated method has stability indicating potential that needs to be further studied.

5

A validated new RP-HPLC method for simultaneous determination of amoxicillin, ampicillin and cloxacillin in pharmaceutical formulations

Gebretsadik Hailekiros, Kahsay Getu, Eticha Tadele, Gebretsadikan Tesfamichael

Acta Chromatographica

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2023

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Vol. 35, no. 2

193--203

EN

As per the World Health Organization, 10% of medicines in low- and middle-income nations are of poor quality and pose a huge public health risk. The development and implementation of cost-effective, efficient and quick analytical methods to control the quality of these medicines is one of the immediate strategies to avoid such a situation. Hence, the main goal of this study was to develop and validate a simple, specific and precise new RP–HPLC method for simultaneous analysis of amoxicillin, ampicillin and cloxacillin in pharmaceutical formulations. The chromatographic analysis was achieved using Shodex C18 (250 × 4.6 mm, 5 μm) column with UV detection at 225 nm. The mobile phase was a gradient mixture of 30 mM phosphate buffer, pH 4.0 (mobile phase A) and acetonitrile (mobile phase B). Efficient separation of the three drugs was obtained using the final optimized chromatographic conditions. The developed method was validated for its specificity, linearity, precision, accuracy and robustness as per the ICH guidelines. The validation results showed that the method was specific, linear, precise, accurate and robust for the simultaneous determination of the three drugs. The developed method was applied to determine the content of the three drugs in pharmaceutical formulations. The assay results of the preparations showed that their drug content was within the pharmacopeial limit stipulated for each drug product. It can be concluded that the proposed method is suitable for simultaneous determination of amoxicillin, ampicillin and cloxacillin in pharmaceutical formulations in industries and regulatory laboratories.

6

A simple HPLC method containing greener modifier and slighter temperature elevated for simultaneous determination of three statin drugs in tablets

Alshitari Wael, Al-Shehri Fatimah, Abd El-Hady Deia, Albishri Hassan M.

Acta Chromatographica

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2022

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Vol. 34, no. 2

210--215

EN

Statins drugs are thought to be among the most prescribed drugs worldwide for the treatment of hypercholesterolaemia. A simple and reliable RP-HPLC method has been successfully employed for simultaneously separating and qualifying three statin drugs including atorvastatin, rosuvastatin and simvastatin in pharmaceutical tablets. The optimal conditions were mobile phase 50:50 (v/v) (formic acid pH 2.50: ETOH), column temperature 40.00 °C, detection wavelength 238.00 nm, and flow rate 1.00 mL/min. The proposed method has been validated based on the ICH guidelines in terms of linearity, precision, accuracy, and limit of detection and limit of quantification. The linear range investigated 2.0–80.0, 4.0–100.00, and 12.00–120.00 µg/mL for rosuvastatin, atorvastatin and simvastatin respectively with coefficients of determination (R2) within the range of 0.9993–0.9995. The LOD and LOQ for rosuvastatin, atorvastatin and simvastatin were (1.57, 4.76 µg/mL), (1.87, 5.66 µg/mL), (3.46, 10.49 µg/mL) respectively. In addition, in order to evaluate the feasibility of the method developed, it was employed towards the quantification of the pharmaceutical tablets for the analytes investigated and excellent recovery was obtained.

7

Development and validation of reversed phase high performance liquid chromatography (RP-HPLC) for quantification of captopril in rabbit plasma

Rasool Muhammad Fawad, Qureshi Umbreen Fatima, Ranjha Nazar Muhammad, Imran Imran, Nisa Mouqadus Un, Majeed Abdul

Acta Chromatographica

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2021

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Vol. 33, no. 4

315--321

EN

Th accurate rapid, simple and selective reversed phase high performance liquid chromatography (RPHPLC) has been established and validated for the determination of captopril (CAP). Chromatographic separation was accomplished using prepacked ODSI C18 column (250 mm 3 4.6 mm with 5 mm particle size) in isocratic mode, with mobile phase consisting of water: acetonitrile (60:40 v/v), pH adjusted to 2.5 by using 85% orthophosphoric acid at a flow rate of 1 mL/min and UV detection was performed at 203 nm. RP-HPLC method used for the analysis of CAP in mobile phase and rabbit plasma was established and validated as per ICH-guidelines. It was carried out on a well-defined chromatographic peak of CAP was established with a retention time of 4.9 min and tailing factor of 1.871. The liquid–liquid extraction method was used for extraction of CAP from the plasma. Excellent linearity (R2 = 0.999) was shown over range 3.125–100 mg/mL with mean percentage recoveries ranges from 97 to 100.6%. Parameters of precision and accuracy of the developed method meet the established criteria. Intra and inter-day precision (% relative standard deviation) study was also performed which was less than 2% which indicate good reproducibility of the method. The limit of detection (LOD) and quantification for the CAP in plasma were 3.10 and 9.13 ng/mL respectively. The method was suitably validated and successfully applied to the determination of CAP in rabbit plasma samples.

8

Rapid RP-HPLC method for simultaneous estimation of metformin, pioglitazone, and glimepiride in human plasma

Sebaiy Mahmoud M., El-Adl Sobhy M., Baraka Mohmed M., Hassan Amira A.

Acta Chromatographica

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2020

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Vol. 32, no. 1

16--21

EN

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed for rapid and simultaneous separation and estimation of 3 antidiabetic drugs, namely, metformin, pioglitazone, and glimepiride, in human plasma within 3 min. Separation was carried out on a MAGELLEN 5U C18 (5 μm, 150 mm × 4.60 mm) using a mobile phase of MeOH–0.025 M KH2PO4 adjusted to pH 3.20 using ortho-phosphoric acid (85:15, v/v) at ambient temperature. The flow rate was 1 mL/min, and the maximum absorption was measured at 235 nm. The retention time of metformin, pioglitazone, and glimepiride was noted to be 1.24, 2.32, and 2.77 min, respectively, indicating a very short analysis time compared to that of other reported methods. Also, limits of detection were reported to be 0.05, 0.26, and 0.10 μg/mL for metformin, pioglitazone, and glimepiride, respectively, showing a high degree of method sensitivity. The method was then validated according to the FDA guidelines for the determination of the three drugs clinically in human plasma, in particular, regarding pharmacokinetic and bioequivalence simulation studies.

9

HPLC–DAD method for investigating pantoprazole for its stress-dependent degradation by photolysis and oxidation

Bratty Mohammad, Thangavel Neelaveni, Peraman Ramalingam, Kumar Vinod, Reddy Padmanabha, Nagappan Krishna Veni, Hazmi Hassan Al

Acta Chromatographica

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2020

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Vol. 32, no. 4

247--255

EN

A reversed-phased high-performance liquid chromatography–diode-array detection (HPLC–DAD) method has been developed for investigating the stress-dependent degradation of pantoprazole (PTZ) by a photolytic and oxidative mechanism. The developed method separated PTZ from its degradation products on a C18 column with a mobile phase consisted of methanol and water (60:40, v/v; pH 3.0) at a flow rate of 1 mL/min. The linear regression coefficient of 0.9995 was obtained for a concentration range from 5 to 25 μg/mL. The % relative standard deviation for repeatability and intermediate precision were below 0.5% and 1.5%, respectively, while the sensitivity of the method was demonstrated by a limit of detection value of 0.25 μg/mL. The stress sample analyses for PTZ results revealed the formation of a total of 18 degradation products, and out of them, 9 degradation products were common for both photolytic and oxidative degradations. Further, the oxidation by azobisisobutyronitrile produced the highest number of degradation products (11 impurities), 3 of which are more hydrophobic than PTZ. In photolytic degradation, 8 and 7 degradation products were observed with UV radiation and sunlight exposure, respectively. Furthermore, the degradation of pantoprazole sodium injection formulation was carried out under the same stress conditions, and it revealed the formation of 3 common impurities under both stress conditions, but other impurities were not detected in the formulations. Finally, 3 common impurities formed in formulations of PTZ injections, viz., sulfone, N-oxide, and N-oxide sulfone impurities, were identified by spike analyses.

10

Development and validation of stability-indicating RP-HPLC method for the simultaneous determination of tizanidine HCL and meloxicam in rabbit's plasma

Zaman Muhammad, Hanif Muhammad, Khan Najm Ul Hassan, Mahmood Asif, Qaisar Muhammad Naeem, Ali Huma

Acta Chromatographica

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2019

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Vol. 31, no. 3

173--178

EN

High-performance liquid chromatography (HPLC) is a widely used technique for the simultaneous detection and quantification of different drugs. The purpose of the current study was to develop a simple and cost-effective reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of tizanidine (TZN) HCl and meloxicam (MLX) in rabbit's plasma. Assay of TZN and MLX was performed after extraction of drug from plasma by liquid–liquid extraction technique using methanol and diethyl ether as protein precipitants. Isocratic elution was performed in a Kromasil® C18 column (dimension, 250 × 4.60 mm; particle size, 5 μm) with mobile phase consisting of methanol–water (8:2). Orthophosphoric acid was used to adjust the pH of the mobile phase 3.0, and detection was done at 228 nm. Flow rate was 0.8 mL/min with ambient temperature and average operating pressure of 1400 psig. Retention time of TZN was 2.612 min and that of MLX was 6.960 min with a resolution of 3.18. Both drugs showed satisfactory linearity in the range of 10 to 50 ng/mL with correlation coefficients (R2) of 0.9989 and 0.9972 for TZN and MLX, respectively. The developed method was validated successfully for linearity, system suitability, intra-day and inter-day accuracy, and precision, robustness, and specificity following International Conference on Harmonization (ICH) guidelines. Conclusively, a precise, stable, reproducible, economical, and suitable method for estimation of pharmacokinetic evaluation was developed and validated.

11

Development and validation of a new isocratic RP-HPLC method for simultaneous determination of sodium metabisulfite and sodium benzoate in pharmaceutical formulation

Ivković B., Brborić J., Dobričić V., Čudina O.

Acta Chromatographica

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2019

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Vol. 31, no. 2

133--137

EN

A simple and convenient reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous separation, identification, and determination of sodium metabisulfite and sodium benzoate in pharmaceutical formulation has been developed and validated. Chromatographic separation was achieved on RP column Zorbax Extend C-18 (150 × 4.6 mm i.d., 3.5 μm particles), and mixture of 0.1% phosphoric acid and acetonitrile in the ratio 62:38 (v/v) was used as a mobile phase. The flow rate was set at 1.0 mL/min with detection wavelength of 275 nm. The method was successfully validated according to International Conference on Harmonization (ICH) guidelines acceptance criteria. The method is selective, as no interferences were observed at retention times corresponding to these analytes. Results of regression analyses (r) and statistical insignificance of calibration curve intercepts (p) proved linearity of the method in defined concentration ranges for sodium metabisulfite and sodium benzoate (0.05–0.15 mg/mL). Relative standard deviations calculated for both analytes in precision testing were below the limits defined for active pharmaceutical ingredients (analysis repeatability: <2%; intermediate precision: <3%). Recovery values were between 98.16% and 101.94%. According to results of robustness testing, chromatographic parameters are not significantly influenced by small variation of acetonitrile content in mobile phase, column temperature, and flow rate. Finally, the method was applied for quantitative determination of investigated preservatives in real sample analysis.

12

Simultaneous quantitative determination of active components in Tetrastigma hemsleyanum by RP-HPLC coupled with diode array detection

Zhu Y., Yin Q., Du P., Huang S., Yang Y.

Acta Chromatographica

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2018

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Vol. 30, no. 3

186--190

EN

An effective, reliable, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) with diode array detector (DAD) method was investigated for simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in Tetrastigma hemsleyanum. The chromatographic separation of the four compounds was carried out on a Welchrom ODS column (4.6 mm × 250 mm, 5 μm) by gradient elution with phosphoric acid (H3PO4) aqueous solution (0.4%)–methanol as the mobile phase, at the temperature of 30 °C and a flow rate of 1.0 mL/min. The detection wavelength was set at 270 nm. Under optimum conditions, the baseline separation of these four compounds can be performed within 30 min. The developed method was validated in terms of detection limit, quantification limit, linearity, precision, and recovery tests. Eventually, the established HPLC–DAD method was successfully applied to the simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in the extract of herb T. hemsleyanum.

13

Development and validation of high-performance liquid chromatography method for determination of some pesticide residues in table grape

Velkoska-Markovska L., Petanovska-Ilievska B., Jankulovska M. S., Ilievski U.

Acta Chromatographica

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2018

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Vol. 30, no. 4

250--254

EN

This study presents the development and validation of a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of captan, folpet, and metalaxyl residues in table grape samples with ultraviolet–diode array detection (UV–DAD). Successful separation and quantitative determination of analytes was carried out on LiChrospher 60 RP-select B (250 × 4 mm, 5 μm) analytical column. Mixture of acetonitrile–0.1% formic acid in water (65:35, v/v) was used as a mobile phase, with flow rate of 1 mL/min, constant column temperature at 25 °C, and UV detection at 220 nm. The target residues were extracted with acetone by ultrasonication, followed by a cleanup using liquid–liquid extraction (LLE) and solid-phase extraction (SPE). The obtained values for multiple correlation coefficients (R2 > 0.90), relative standard deviation (RSD) of retention times, peak areas and heights (RSD ≤ 2.25%), and recoveries ranging from 90.55% to 105.40%, with RSD of 0.02% to 5.37%, revealed that the developed method has a good linearity, precision, and accuracy for all analytes. Hence, it is suitable for routine determination of investigated fungicides in table grape samples.

14

Zastosowanie wysokosprawnej kolumnowej chromatografii cieczowej w odwróconych układach faz (RP-HPLC) do rozdzielania i wstępnej identyfikacji składników hydrofilowych mieszanin po hydrolizie zasadowej biomasy lignocelulozowej (BMLC)

Nowak P., Łukajtis R., Kamiński M.

Camera Separatoria

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2017

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Vol. 9, No. 2

131--154

PL

Wzrost konsumpcji paliw kopalnych, ich pozyskiwanie oraz eksploatacja niesie ze sobą wiele zagrożeń dla środowiska, dlatego alternatywnym źródłem energii stają się biopaliwa, w tym bio-wodór pozyskiwany w konwersji biomasy ligno-celulozowej, która poddawana jest obróbce wstępnej. Najczęściej wykorzystywaną metodą obróbki wstępnej jest hydroliza alkaliczna, podczas której powstaję bardzo dużo produktów ubocznych, nieprzydatnych do wytwarzania paliw, szczególnie powstałych z hydrolizy ligniny. Najczęściej wykorzystywaną techniką identyfikacji i oznaczania składu hydrolizatów biomasy ligno-celulozowej jest chromatografia cieczowa realizowana w różnych układach faz oraz z wykorzystaniem elucji gradientowej. W przypadku badania hydrolizatów zawierających hydrofobowe składniki, najbardziej korzystne wydają się warunki odwróconych układów faz – RP-HPLC. W niniejszej pracy porównano dwie metodyki wysokosprawnej kolumnowej chromatografii cieczowej w odwróconych układach faz (RP-HPLC) do rozdzielania i wstępnej identyfikacji składników hydrofilowych mieszanin po hydrolizie zasadowej biomasy lignocelulozowej, w celu optymalizacji procesu konwersji biomasy ligno-celulozowej (BMLC) do uzyskania najlepszej efektywności procesu hydrolizy. Wyniki tych badań powinny doprowadzić w przyszłości do procedur pozyskania ubocznych produktów, powstających podczas procesu hydrolizy BMLC, przydatnych użytkowo. Konieczne będą badania uzupełniające, wykonywane w warunkach dwu-wymiarowej elucyjnej gradientowej kolumnowej wysokosprawnej chromatografii cieczowej, z uwzględnieniem spektrometrii Mas (MS), oprócz detektora spektrofotometrycznego z detektorem typu DAD (Diode Array) - 2D-Grad-HPLC-UV-VIS-DAD / MS.

EN

The increase in the consumption of fossil fuels, their acquisition and exploitation carries a lot of threats to the environment, therefore an alternative source of energy are biofuels, including biohydrogen obtained in the conversion of ligno-cellulosic biomass, which undergoes pre-treatment. The most frequently used method of pre-treatment is alkaline hydrolysis, during which a lot of by-products are generated, unsuitable for the production of fuels, especially those resulting from hydrolysis of lignin. The most commonly used technique for identifying and determining the composition of lignocellulose biomass hydrolysates is liquid chromatography carried out in various phase systems and using gradient elution. In the case of testing hydrolysates containing hydrophobic components, the conditions of reversed phase systems - RP-HPLC seem to be most favorable. This paper compares two methods of high performance reverse phase column chromatography (RP-HPLC) for the separation and initial identification of components of hydrophilic mixtures after basic hydrolysis of lignocellulose biomass, in order to optimize the process of converting lignocellulose biomass (BMLC) to the best the effectiveness of the hydrolysis process. The results of these tests should lead in the future to procedures for obtaining by-products of BMLC hydrolysis which are useful for use. Supplementary tests will be required, performed in two-dimensional, elution, gradient, columnar high performance liquid chromatography, including Mas (MS) spectrometry, in addition to a spectrophotometric detector with a DAD detector (Diode Array) - 2D-Grad-HPLC-UV-VISDAD / MS.

15

Stress testing of linaclotide: Development of a validated stability-indicating RP-HPLC method

Pyla S. R. M., Sahu P. K., Srinivas K.

Acta Chromatographica

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2017

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Vol. 29, no. 2

207--217

EN

Linaclotide, a first-in-class guanylate cyclase-C agonist, was recently approved by US Food and Drug Administration (FDA) as a promising pharmacotherapy for the management of constipation-predominant irritable bowel syndrome (IBS). In this communication, we present a novel stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of linaclotide along with its degradation products. During the International Conference on Harmonization (ICH) prescribed stress study, linaclotide was found susceptible to degrade under hydrolytic (acid and base) and oxidative (peroxide) conditions. The separation of the degradants from the analyte was achieved on a Zorbax Eclipse XDB C8 Column (250 mm × 4.6 mm, 5 μm) using 0.01 N potassium dihydrogen orthophosphate buffer and acetonitrile (80:20 v/v) as mobile phase at a flow rate of 1.00 mL min−1 at column temperature of 40 °C. The detection of the column effluents was realized on a photodiode array detector set at 220 nm. Under the above optimal condition, the method was validated with respect to specificity, linearity, range, precision, robustness, and sensitivity in compliance to the regulatory requirements.

16

Development and validation of a reversed-phase HPLC method for simultaneous determination of doxazosin mesylate and finasteride

Krotz D., Mengesha A. E.

Acta Chromatographica

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2017

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Vol. 29, no. 3

309--324

EN

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of doxazosin mesylate (DOX) and finasteride (FIN) in bulk powders and pharmaceutical formulations. The compounds were separated on a Pinnacle II C18 column (250 × 4.6 mm i.d.; particle size, 5 μm) with an isocratic mobile phase at a flow rate of 1.0 mL min−1. The mobile phase was a mixture of 25 mM ammonium acetate and acetonitrile in the ratio of 50:50 %v/v. The pH of the buffer was adjusted to 4.0 ± 0.05 with glacial acetic acid. The detection was performed at 230 nm. The total chromatographic analysis time per sample was 15 min with DOX and FIN eluting at 3.9 and 7.2 min, respectively. The accuracy, precision, specificity, linearity, and sensitivity of the method were validated according to the International Conference on Harmonization (ICH) guidelines. The calibration plots were linear (r2 > 0.999) over the concentration range 24.25–291.0 μg mL−1 and 122.5–1470.0 μg mL−1 for DOX and FIN, respectively. The method was used for the simultaneous determination of DOX and FIN in capsules.

17

Determination of selected antiepileptic drugs in mouse brain homogenates by HPLC—DAD

Wróblewski K., Petruczynik A., Radzik I., Czuczwar S. J., Waksmundzka-Hajnos M.

Acta Chromatographica

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2017

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Vol. 29, no. 2

219--234

EN

Three independent reversed phase high-performance liquid chromatography (HPLC) procedures with diode array detection (DAD) for the analysis of carbamazepine (CBZ), topiramate (TPM), and valproic acid (VPA) have been developed in order to determine drug penetration of the blood—brain barrier. Determination of CBZ was performed on C18 column with mobile phases containing methanol (55%, v/v), acetate buffer at pH 3.5 (20%, v/v), double distilled water (25%, v/v), and 0.025 M L−1 diethylamine (DEA). The mobile phase containing acetonitrile and water (8:2, v/v) or acetonitrile and phosphate—citrate buffer at pH 2.6 (1:1), respectively, for analysis of VPA and TPM was applied. Quantification of carbamazepine was performed at 285 nm without extraction procedure before the analysis. Determination of topiramate and valproic acid was performed using precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). FMOC-Cl is a suitable agent, which reacts with both primary and secondary amines and also with acidic groups. Topiramate was determined at 263 nm and valproic acid at 300 nm. The proposed procedures are simple, not time-consuming, and suitable for the determination of investigated compounds in mouse brain homogenates.

18

Simultaneous analysis of adenosine triphosphate and its metabolites in Saccharomyces Cerevisiae using RP-HPLC

Zhu P., Wang S., Wang J., Zhou L., Shi P.

Acta Chromatographica

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2016

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Vol. 28, no. 4

501--511

EN

In order to assess the contribution of adenosine triphosphate and its metabolites to the cellular metabolism process in Saccharomyces cerevisiae, it is very important to simultaneously determine the relative concentrations of ATP and its metabolites. In this study, a fast, simple reversed-phase high-performance liquid chromatography with high selectivity was developed to simultaneously measure adenosine triphosphate and its metabolites (adenosine diphosphate, adenosine monophosphate, and cyclic adenosine monophosphate) in yeast. The method was performed under the gradient grogram, and the detection was monitored at 254 nm. Analysis was achieved within 25 min. The four components can be detected with linear response over the concentration range from 1 to 100 mg L−1 with excellent correlation coefficients (r2) > 0.999. The recovery of the four analytes was 92.9%, 90.4%, 99.1%, and 105.1%, respectively. To demonstrate the good analysis of yeast samples, changes in the four adenine nucleotides levels caused by caloric restriction in yeast were determined. It is expected that the current method may contribute to further metabolomics and system biology investigations of yeast.

19

LC—MS/MS Studies for the Identification and Characterization of Degradation Products of Fenofibrate and Their Degradation Pathways

Mulgund S. V., Anbazhegan S., Gabhe S. Y.

Acta Chromatographica

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2016

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Vol. 28, no. 2

159--173

EN

In the present study, the degradation behavior of Fenofibrate under different International Conference on Harmonization (ICH) suggested conditions was studied. Characterization of degradation products by liquid chromatography–tandem mass spectrometry (LC–MS/MS) studies in solution form was done, and the possible mechanism for the formation of degradants is discussed. Fenofibrate was subjected to different hydrolytic stress conditions and thermal stress condition (in solid form). Successful separation of drug from degradants was achieved on a C18 column using water–acetonitrile (25:75 v/v) as the mobile phase. Other high-performance liquid chromatography (HPLC) parameters were: flow rate, 1 mL min−1; detection wavelength, 286 nm; column temperature, 25 °C; and injection volume, 20 μL. The method was validated for linearity, precision, accuracy, robustness, and specificity and was stability-indicating one, based on the specificity studies. The drug degraded under acidic, basic, and oxidative hydrolytic stress while it was relatively stable towards neutral hydrolysis and thermal stress. The stressed samples were subjected to LC–MS/MS analysis. On the basis of spectral data, the structures of four degradation products and one interaction product were suggested. Degradation products were characterized to be isopropyl acetate, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propanoic acid, 4-hydroxy benzoic acid, and benzoic acid. The structure of one interaction product was proposed as methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate.

20

Fast and Sensitive RP-HPLC—Fluorescence Method for the Quantitative Analysis of Moxifloxacin in Rat Plasma and Its Application to a Preclinical Pharmacokinetic Study

Hurtado F. K., Kaiser M., Tasso L., Costa T.

Acta Chromatographica

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2016

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Vol. 28, no. 2

175--191

EN

A fast, simple, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and fully validated for the determination of moxifloxacin (MXF) in rat plasma. MXF and gatifloxacin (internal standard, I.S.) were extracted from plasma by single-step protein precipitation with acidified acetonitrile. Chromatographic separation was accomplished in less than 8 min on an Atlantis ® T3 column with 0.4% aqueous triethylamine–methanol–acetonitrile (60:35:5, v/v/v) solution as mobile phase. Detection was achieved by fluorescence (λexcitation = 295 nm, λemission = 500 nm), and the calibration curves were found to be linear over the plasma concentration range of 10–2,500 ng mL−1 with a mean correlation coefficient (r) of 0.9946 (n = 6). The intra- and inter-assay imprecision (% CV) was less than 2.4 and 3.3%, respectively, and the accuracy was >90%. The mean extraction recoveries for MXF and I.S. from plasma were 77 and 82%, respectively. The method was also validated for specificity, sensitivity, and stability; all the results were within the acceptable range. The proposed method was then successfully applied to the quantitative analysis of MXF in rat plasma samples, being a valuable and high-throughput assay to support ongoing pharmacokinetic studies on this promising anti-infective agent.