Design of Experiments (DoE)-based approach for improvement of dry mixing processes in the production of low-dose Alprazolam tablets using Raman spectroscopy for content uniformity monitoring

A low-dose tablet formulation, containing a potent Benzodiazepine derivative Alprazolam was developed, considering the achievement of appropriate content uniformity of the active substance in powder blends and tablets as a major challenge. Two different types of lactose monohydrate (Tablettose 80 and Granulac 200) and two different types of dry mixing processes (high-shear mixing and "in bulk" mixing) were employed. To evaluate the influence of the variables (mixing speed, mixing time, filling level of the high-shear and cube mixer, lactose monohydrate type) and their interactions upon the response (content uniformity of Alprazolam in the powder blends), a Factorial 2 4 design (with 4 factors at 2 levels in 1 block) was generated for each type of mixer. For high-shear dry mixing the Response Surface, D-optimal Factorial 2 4 design (with 2 replications and 31 experiments) was used, while for the "in bulk" dry mixing the Response Surface, Central Composite Factorial 2 4 design (with 34 experiments) was used. The process parameters for the high-shear mixer were varied within the following ranges: filling level of 70-100%, impeller mixing speed of 50-300 rpm and mixing time of 2-10 minutes. For the cube mixer the following process parameter ranges were employed: filling level of 30-60%, mixing speed of 20-390 rpm and mixing time of 2-10 minutes. Raman spectroscopy in conjunction with a validated Partial Least Square (PLS) regression model was used as a Process Analytical Technology (PAT) tool for Alprazolam content determination and content uniformity monitoring. The DoE model was further employed to optimize the powder blending process in regard to the achievement of appropriate Alprazolam content uniformity using high-shear mixing and Tabletosse 80 as filler. The desirability function revealed that the following process parameters: a mixing time of 2 minutes, a mixing speed of 300 rpm and a 70% filling level of the mixer would produce powder blends with the lowest variability in Alprazolam content. The three independent lab batches of low-dose Alprazolam tablets, produced with high-shear mixing using these process parameters, conformed to the requirements of the European Pharmacopoeia for content (assay) of Alprazolam and uniformity of the dosage units

Development and validation of a fast and simple HPLC method for the simultaneous determination of bisoprolol and enalapril in dosage form

Aim. We aimed to develop and validate fast, simple, accurate, robust and rugged chromatographic method for simultaneous determination of bisoprolol fumarate and enalaril maleate in solid pharmaceutical dosage form, with using chaotropic strong chaotropic perchlorate anions in composition of mobile phase. Materials and methods. Fast simple HPLC method for simultaneous determination of bisoprolol and enalapril in solid pharmaceutical dosage forms was developed, with perfect peak symmetries eluting at 4.7 and 5.2 miutes, with mobile phase composed of methanol and diluted perchloric acid pumped with 1ml/min on Zorbax Rx C8 250x4.6mm, 5um column thermostated at 42 °C, and monitored UV signal at 214nm. Mobile phase was composed of 55%methanol and 45% perchloric acid (0.07%v/v). Results. Usage of presence of perchloric anions showed very useful role in peak shape and chromatogram view, due to distinguished chaotropic characteristics of perchlorate anions on molecules containing nitrogen atoms in molecular structures of analytes, in acidic pH environment. Linearity was examined and proven at different concentration levels in the range of working concentration of bisoprolol (20–200 ug/ml) and enalapril (20–200 ug/ml). The methods achieved very good validation parameters, with determined LOQ about 0.032 mg/ml and LOD about 0.003 mg/ml for bisoprolol, and LOQ about 0.045 mg/ml and LOD 0.005 mg/ml for enalapril. The high value of recoveries obtained for bisoprolol and enalapril indicates that the proposed method was found to be accurate. Conclusion. A new fast and simple, but selective, accurate, precise and robust HPLC method for simultaneous determination of bisoprolol and enalapril in tablets was developed and many possible variations of the same were suggested. The developed method for the simultaneous quantification of bisoprolol and enalapril from solid dosage formulations offers simplicity essential for quality control of a large number of samples in short time intervals, which is necessary for routine analysis

Preliminary investigation of the possibility for implementation of modified pharmacopoeial HPLC methods for quality control of metronidazole and ciprofloxacin in medicinal products used in veterinary medicine

Quality control of veterinary medicine products containing two different frequently used antibiotics metronidazole and ciprofloxacin hydrochloride, was considered and performed, using modified pharmacopoeial HPLC methods. Three different HPLC systems were used: Varian ProStar, Perkin Elmer Series and UPLC Shimadzu Prominence XR. The chromatographic columns used were LiChropher RP Select B 75 mm x 4 mm with 5 μm particles and Discovery C18 100 mm x 4,6 mm with 5 μm particles. Chromatographic methods used for both analytes were compendial, with minor modifications made for experimental purposes. Minor modifications of the pharmacopoeia prescribed chromatographic conditions, in both cases, led to better chromatographic parameters, good resolution and shorter analysis times. Optimized methods can be used for: determination of metronidazole in gel formulation, for its simultaneous quantification with preservatives present in the formulation and even for identification and quantification of its specified impurity, 2-methyl-5-nitroimidazole; determination of ciprofloxacin hydrochloride in film coated tablets and eye drops and identification and quantification of its specified impurities. These slightly modified and optimized pharmacopoeial methods for quality control of metronidazole and ciprofloxacin dosage forms used in veterinary medicine can be successfully applied in laboratories for quality control of veterinary medicines

Concepts for New Rapid Simple HPLC Method for Quantification of Fosfomycin Trometamol in Pharmaceutical Dosage Forms with Direct UV Detection

Two different concepts for developing direct HPLC-UV methods for quantifying fosfomycin trometamol were developed without any derivatization and modification of the analyte. In the first concept, without the use of alkylamines as ion-pairs in the mobile phase, by using cyanopropyl CN and a strong anion-exchanger column, we investigated the possibility of their highly polar and anion-exchanging forces and mechanisms to retain, separate and detect trometamol without the help of additional agents or modifiers. In the second concept, the most frequent reversed-phase C18 columns with different characteristics and vendors were tested in combination with different length-based alkylamines with 3–10 C atoms in their chains. In our research, we found that the ion-pairing of fosfomycin with 6–10 C-atom-based alkyl-length of aliphatic chains manifested the most appropriate strength of interactions between alkyl-paired trometamol molecules and octadecylsilane or C18 bonded RP column to achieve optimal retention, selectivity and peak shape on chromatograms, with the possibility for the fine-tuning of elution time. The simplicity of our method concept omits the need for expensive and sophisticated columns like HILIC, C30 graphite carbon, and mixed-mode-based columns for easier retaining, separation, and determination of fosfomycin, and for its quantification purposes, especially in high-throughput analyses in regular quality-control laboratories

Concepts for New Rapid Simple HPLC Method for Quantification of Fosfomycin Trometamol in Pharmaceutical Dosage Forms with Direct UV Detection

Two different concepts for developing direct HPLC-UV methods for quantifying fosfomycin trometamol were developed without any derivatization and modification of the analyte. In the first concept, without the use of alkylamines as ion-pairs in the mobile phase, by using cyanopropyl CN and a strong anion-exchanger column, we investigated the possibility of their highly polar and anion-exchanging forces and mechanisms to retain, separate and detect trometamol without the help of additional agents or modifiers. In the second concept, the most frequent reversed-phase C18 columns with different characteristics and vendors were tested in combination with different length-based alkylamines with 3–10 C atoms in their chains. In our research, we found that the ion-pairing of fosfomycin with 6–10 C-atom-based alkyl-length of aliphatic chains manifested the most appropriate strength of interactions between alkyl-paired trometamol molecules and octadecylsilane or C18 bonded RP column to achieve optimal retention, selectivity and peak shape on chromatograms, with the possibility for the fine-tuning of elution time. The simplicity of our method concept omits the need for expensive and sophisticated columns like HILIC, C30 graphite carbon, and mixed-mode-based columns for easier retaining, separation, and determination of fosfomycin, and for its quantification purposes, especially in high-throughput analyses in regular quality-control laboratories

Novel HPLC-UV method for simultaneous determination of valsartan and atenolol in fixed dosage form; Study of green profile assessment

Aim of this work was to develop the first simple, rapid, green, economical and selective HPLC method for simultaneous quantification of the cited drugs in their challenging binary mixture. The work was motivated by the global trends towards sustainable chemistry in designing eco-friendly mobile system without affecting the analysis parameters. The proposed method was subjected to a greenness profiles using some metrics as Eco-scale. Materials and methods. This was accomplished under the following chromatographic conditions: HPLC column Discovery C18 (4.6 mm i.d. × 150 mm, 5 μm), column temperature 30 °C, flow rate 1.0 mL/min, mobile phase composed of 20% acetonitrile, 80% of 0.16% ammonium acetate and 0.2% of 1.5 M tetramethylammonium hydroxide (V/V) and signal monitoring at a wavelength of 225 nm and 237 nm. Results. A conventional mixture of acetonitrile and 0.16% ammonium acetate was tried in different ratios, but the drugs were not well separated. The shortest aliphatic chain cationic ion pair reagent tetramethylammonium hydroxide should not be exchanged with other type similar with this, like tetramethylammonium hydrogen sulfate, it did not work to our experiments. Increasing salt concentration, ammonium acetate, more than 0.2%, pushes the peak of atenolol closer to dead volume, which is negative. Atenolol in their methods for multicomponent mixtures elutes in dead volume, or when retained longer, much stronger, hydrophobic mobile phase should be used if valsartan should be seen in same chromatogram at dissent time. The 237 nm can be chosen as compromise signal for nearly equal peaks height with high sensitivity is not essential. The 225 nm signal shows much higher sensitivity for atenolol and less increase for valsartan peaks, which can be used when higher sensitivities will be essential. Linearity was examined and proven at different concentration levels in the range of working concentration of valsartan (0.16–0.96 mg/mL) and atenolol (0.2–1.20 mg/mL). The high value of recoveries obtained for valsartan and atenolol indicates that the proposed method was found to be accurate. The results of proposed method found to be an excellent green analysis with a score of 84. Conclusion. A new fast, simple and green, but selective, accurate, precise and robust HPLC-UV method for simultaneous determination of valsartan and atenolol in newly formulated dosage form was developed and many possible variations of the same were suggested. The developed method for the simultaneous quantification of valsartan and atenolol in their challenging binary mixture offers simplicity essential for quality control of a large number of samples in short time intervals, which is necessary for routine analysis. The method was subjected to greenness profile assessment

Method development for the quantitative determination of captopril from Caco-2 cell monolayers by using LC-MS/MS

Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers

Concepts for a New Rapid and Simple HPLC Method for Simultaneous Determination of Metoprolol and Meldonium in Pharmaceutical Dosage Forms

Simultaneous determination of the tandem of drugs, like meldonium and metoprolol, with enormous polarity differences between them, requires thorough research and careful selection of chromatographic conditions. The three different CN-cyano groups with link-based particle columns, LiChrospher CN, Waters Spherisorb CNRP, Zorbax CN SB stationary phases, were tested, in an isocratic elution system, with a running mobile phase containing various concepts of composition contents. They were first with buffering salts which included acetonitrile and ammonium phosphate in one group, and then without buffering salts but with diluted acids, composed of acetonitrile and diluted acids as the second group. We can conclude that the most optimal concepts, in terms of expressiveness and environmental friendliness, were concepts using of column Zorbax CN SB (4.6 mm i.d. × 250 mm, 5 μm) and mobile phase ACN—0.15% NH4H2PO4 (50:50 and 60:40, v/v). There are very poor available data about ideas and usable information about the development of methods for simultaneous determination of these two active substances with polarity differences between them. We suggest that our work offered detailed and successful solutions for the mentioned aim using less sophisticated equipment for quality control and a lab for routine manufacturing control

Development of a Novel, Fast, Simple HPLC Method for Determination of Atorvastatin and its Impurities in Tablets

Our main target and concept was to develop a method for the determination of the most prescribed antilipemic drug, atorvastatin, together with its related substances, with a single sample preparation and during a single chromatographic run, in the shortest possible period of time, with the lowest possible mobile phase consumption. A new rapid, simple chromatographic method for the determination of atorvastatin and its main specified impurities was developed, using different chromatographic columns. With this new concept of a mobile phase and a powerful core–shell, or a superficially porous silica-based column, satisfactory results for targeted parameters, such as critical peak resolution, run time length, and column backpressure, were achieved. The analysis is performed within a run duration of less than 15 min, which is about six times shorter than the official European Pharmacopoeia method. The chromatogram performances suggests that the method limit of quantification (LOQ) can be about 7 times lower, and the limit of detection (LOD) about 20 times lower, using an injection volume of only 2 µl. This was confirmed by the performed method validation in accordance with the International Conference on Harmonization (ICH) guideline for the validation of analytical procedures Q2(R1), where the selectivity, linearity, accuracy, precision, limit of quantification, and limit of detection were tested and confirmed

Застосування солей хаотропних аніонів в розробці верх методики визначення мельдонію в лікарських формах

The aim of the work was to create an approach for the development of HPLC methods for the determination of meldonium in dosage forms with the usage of salts of chaotropic anions in mobile phases. Material and methods. Analytical equipment: Shimadzu UPLC system LC-40 PDA; Shimadzu Nexera-i LC-2040C 3D-Plus, controlled by software Lab Solution version 5.97, electronic laboratory balance RAD WAG AS 200/C, pH-meter I-160MI. Meldonium dihydrate (purity 99.3 %) was purchased from Sigma-Aldrich (Switzerland), and Vasopro capsules 500 mg were purchased from a local pharmacy. Chromatographic conditions: Agilent Zorbax C-18 SB 150 mm x 4.6 mm 3.5 μm column was used (Agilent Technologies, USA). Mobile phases: 1) 0.25 % KPF6 w/v – 0.1 % v/v 85 %H3PO4 95 % – 5 % ACN, 2) 0.3 % bis-(trifluoromethane)sulfonimide lithium salt 97 % w/v – 0.1 %v/v 85 % H3PO4 80 % – 20 % acetonitrile. Flow rate - 1mL/min, T=32 °C, detection UV=at 4 channels - 190 nm, 195 nm, 200 nm, 205 nm. Results and discussion. We have proposed two approaches using two different salts of chaotropic anions - potassium hexafluorophosphate and bis-(trifluoromethane)sulfonimide lithium salt – for the HPLC method development. The chaotropic effects of these anions toward meldonium strongly influenced the analyte migratory behaviour. Both mobile phases involved, in addition to the use of a chaotrope, also the use of acetonitrile and pH adjustment with 0.1 % v/v 85 % H3PO4 solution. The detection wavelength (190 nm, 195 nm, 200 nm, 205 nm) was selected experimentally. The results were obtained for 8 concepts. Parameters of the chromatographic system confirm the conclusions and results of this investigation for the influence of chaotropic salts on N-containing molecules in an acidic pH medium, by increasing their retentivity and improving peak shape and uniformity homogeneity, even on the column without end-capping and base-deactivating. Validation of the analytical method was carried out following the requirements of SPhU. Conclusions. HPLC methods for the determination of meldonium in dosage forms have been developed, using positive impacts of chaotropic salts on the molecules containing N-atoms in their molecule on their retentions and peak symmetries on the chromatogram. The validation of the analytical methods showed their suitability for pharmaceutical analysisМетою роботи було створення підходів до розробки ВЕРХ методик визначення мельдонію в лікарських формах з використанням солей хаотропних аніонів у складі рухомих фаз. Матеріали і методи. Аналітичне обладнання: Shimadzu UPLC system LC-40 PDA; Shimadzu Nexera-i LC-2040C 3D-Plus, що керується програмним забезпеченням Lab Solution версія 5.97, ваги лабораторні електронні RAD WAG AS 200/C, pH-метр И-160МИ. Мельдонію дигідрат (чистота 99,3 %) закуплено у компанії Sigma-Aldrich (Швейцарія), «Вазопро» капсули по 500 мг закупляли у місцевій аптеці. Хроматографічні умови: використовували колонку Agilent Zorbax C-18 SB 150 мм x 4.6 мм 3.5 мкм (Agilent Technologies, США). Рухомі фази: 1) 0.25 % KPF6 w/v – 0.1 % w/v 85 % H3PO4 95 % – 5 % ACN, 2) 0.3 % біс-(трифторметан)сульфонімід літієвої солі 97 % w/v – 0.1 % v/v 85 % H3PO4 80 % – 20 % ацетонітрилу. Швидкість потоку – 1 мл/хв, T=32 °C, детектування УФ на 4 довжинах хвиль - 190 нм, 195 нм, 200 нм, 205 нм.Результати і обговорення. Для розробки методики ВЕРХ ми запропонували два підходи з використанням двох різних солей хаотропних аніонів – гексафторфосфату калію та біс-(трифторметан)сульфоніміду літієвої солі. Хаотропний вплив цих аніонів на мельдоній сильно вплинув на міграційну поведінку аналіту. Обидві рухомі фази включали, крім використання хаотропу, також використання ацетонітрилу та регулювання рН за допомогою 0.1 % об./об. 85 % розчину H3PO4. Довжини хвиль детектування (190 нм, 195 нм, 200 нм, 205 нм) підбирали експериментально. Результати отримано для 8 концепцій. Параметри хроматографічної системи підтверджують висновки та результати цього дослідження щодо впливу хаотропних солей на N-вмісну молекулу в кислому середовищі pH, збільшуючи їх здатність до утримування та покращуючи форму піку і рiвномiрну одноріднiсть, навіть на колонці без кінцевого закриття та дезактивації основи. Валідацію аналітичної методики проведено відповідно до вимог ДФУ.Висновки. Розроблено ВЕРХ методики визначення мельдонію в лікарських формах з використанням позитивного впливу хаотропних солей на молекули, що містять N-атоми, на їх утримування та симетрії піків на хроматограмі. Валідація аналітичних методик показала їх придатність для цілей фармацевтичного аналізу