Mobile Phase Mixture Research Articles

Algorithm for assessing the completeness of seed treatment of wheat and barley

Goal. To develop a method for the determination of imidacloprid, clothianidin, prothioconazole and tebuconazole in the treatment seeds of wheat and barley.
 Methods. Active substances were determined by thin layer chromatography (TLC). Mathematical-statistical method for evaluate the linear range of the dependence of the area of the chromatographic zone on the amount of active substance was used.
 Results. The determination of pesticides includes a number of basic stage: characteristic the matrix by the ratio of the content of coextracting substances, that interfere with the determination, and the content of pesticides; classification of pesticides by polarity in according to the dipole moment (µ, D); extraction from the analyzed sample by organic solvent with corresponding of the dielectric constant, ε; purification; qualitative and quantitative determination. In the case of the analysis of treatment seeds, in which the amount of pesticides is much greater than coextracting compounds, the stages of classification and purification are excluded from the analysis. The extraction is carried out with ethanol. Qualitative determination of pesticides is carried out on plates with a thin layer of adsorbent (silicagel) in the mobile phase (mixture of hexane and ethanol, ε = 7.49) with using corresponding reagents. Silver ammonia (AgNO3) solution and followed by UV irradiation of the chromatogram is used for identification halogen-containing pesticides (zones of localization of compounds are formed dark spots of reduced silver). Pesticides containing donor atoms of sulfur, nitrogen, oxygen, identify by bromphenol blue (BPB) and subsequent bleaching of the background with an solution of citric acid (zones of localization of compounds are formed blue spots form on a light background). The active substances are identified with the corresponding Rf of the zones localization. Quantitatively of active substances is determined by the calibration dependence of the area of compound’ chromatographic zone on its quantity. For all four compounds, this dependence is linear in the concentration range of 0.20 — 0.80 µg and is described by the regression equation for: imidacloprid y = 6.25x + 5.45; for clothianidine y = 11.65x + 6.70; for prothioconazole y = 28.7x + 2.05; for tebuconazole y = 13.00x + 7.00. In all cases, the correlation between the indicators is estimated as strong (r = 0.99).
 Conclusions The algorithm for assessing the completeness of the treatment of wheat and barley seeds is an express control method and using the thin layer chromatography method allows to determine four active substances in one sample during one analysis with high accuracy (relative error of less than 5% at n = 5, P = 0.95) excluding laborious experiment.

Determination and validation of Tetracycline residues in Poultry by High Performance Liquid Chromatography - Diode Array Detector Technique

The aim of this research is separation and determination of trace Tetracycline residues in Poultry chest, thigh and liver using High Performance Liquid Chromatography - Diode Array Detector (HPLC-DAD), with a mobile phase mixture consisting of acetonitrile: methanol: oxalic acid (0.01M) (25:15:60) and chromatographic column C8. The study was done on 32 live poultry individuals. All individuals were injected in the chest by 1m of Tetracycline standard solutions, then slaughter for analysis throughout four successive days. The injection with 10×103 ppb of Tetracycline showed that the traces of Tetracycline residues (according to the Codex Alimentarius Commission) exceeded the value of the maximum residue limit (MRL = 200 ppb) in the thigh and chest meat at the 1st day and the 1st & 2nd days of slay respectively, and exceeded the value of (MRL = 600 ppb) in the liver at the 3rd and 4th days of slay. limit of detection was LOD = 0.451 ppb, limit of quantification LOQ = 1.502 ppb, and recovery percentages of Tetracycline at a concentration of 200.0 ppb for 20 sample Rec.% = (88.966 - 91.055%), (84.623 - 87.667%), (82.198 - 83.688%) for Poultry chest, thigh and liver respectively with a percentage relative standard deviations (RSD%) of < 1 %.

Chromatographic Quantification of Ivermectin and Pranziquantel in the Tablets Using Stability Indicating RP-HPLC Method

Background: A new stability indicating RP-HPLC based assay method was developed to quantify ivermectin and praziquantel simultaneously and applied effectively to tablets. Methods: The simultaneous assay of ivermectin and praziquantel by RP-HPLC was done using an YMC C18 (250 mm × 4.6 mm, 5 µm) column with a mobile phase mixture of 0.1M disodium hydrogen phosphate (pH 4.5) and acetonitrile (55:45, v/v) using a isocratic flow rate of 1.0 ml/min and measured at 242 nm using photodiode array detector. All parameters were validated following the ICH guiding principles. The method was applied to quantify ivermectin and praziquantel simultaneously in tablets. Results: The retention values of ivermectin and praziquantel were 3.465 min and 4.468 min, respectively. The method’s linearity was found to be 1-3 µg/ml (ivermectin) and 25-75 µg/ml (praziquantel). The limit of detection was 0.010 µg/ml (ivermectin) and 0.046 µg/ml (praziquantel); limit of quantification was 0.033 µg/ml (ivermectin) and 0.155 µg/ml (praziquantel). The percent relative standard deviation of ivermectin and praziquantel was ˂1.0%. The percent assay was 99.51% and 99.20% for ivermectin and praziquantel, respectively. In tablets, the percent recovery of ivermectin and praziquantel was 99.60% and 99.38% with a percent relative standard deviation value of 0.353% and 0.106%, respectively. Stability indicating capability of the method was demonstrated through the stress degradation studies. Conclusion: The developed method was proved to be selective, precise and accurate for the quality control of ivermectin and praziquantel in tablets.

Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale.

Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with sub/supercritical fluid chromatography (SFC) moving toward these new applications beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at the preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein, we overcome these problems by introducing chaotropic effects in SFC separations using a nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3-). In contrast to other salts, HCO3- was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen-deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.

Gas chromatography and liquid chromatography coupled to mass spectrometry for the determination of fluorotelomer olefins, fluorotelomer alcohols, perfluoroalkyl sulfonamides and sulfonamido-ethanols in water

In this work, the suitability of gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the multi-class determination of different families of neutral per- and polyfluoroalkyl substances (PFASs), such as fluorotelomer olefins (FTOs), alcohols (FTOHs) and fluorooctanesulfonamides (FOSAs) and sulfonamido-ethanols (FOSEs), was investigated and compared. Regarding GC–MS, the use of a semi-polar GC column (DB-624, 6%-cyanopropilphenyl 94%-dimethyl polysiloxane) allowed the adequate separation of all the compounds while chemical ionisation (CI) of positive ions as ionisation technique provided the best responses. Concerning UHPLC–MS/MS, atmospheric pressure chemical ionisation (APCI) and photoionisation (APPI) sources allowed the ionisation of all studied neutral PFASs, including FTOs for the first time. High vaporizer temperatures (450 °C) and acetonitrile/water mobile phase mixtures were required to favour the ionisation of FTOs, with adequate ionisation for FTOHs, FOSAs and FOSEs. The chromatographic separation, performed on a totally porous column (Luna C18), allowed the successful separation of the four families of neutral PFASs. After comparing the performance of the studied methods, the highest detectability was achieved using UHPLC–APCI–MS/MS and it was chosen in combination with a solid-phase extraction (SPE) procedure for the analysis of neutral PFASs in water samples. The whole method provided low limits of detection (0.003–6 µg L–1), good precision (RSD < 9%) and trueness (relative error < 10%). The methodology was applied to the analysis of river water samples and the presence of some neutral PFASs were detected (8:2 FTO) and quantified (4:2 FTOH and N-EtFOSA) at low concentration levels (ng L–1).

Determination of Radiochemical Purity of 99mTc-DTPA Using One-System Method of Paper Chromatography

The more efficient and effective quality control techniques for 99mTc-DTPA are needed because 99mTc has a short half-life of around 6.0 hours. We have succeeded in developing a one-system of Thin Layer Chromatography (TLC) for radiochemical purity testing system that is faster and more practical. Two-system method of TLC for radiochemical purity testing uses mobile phase of methyl ethyl ketone indicated as system A and 0.9% sodium chloride solution indicated as system B. One-system method uses the mobile phase of a mixture solution of acetone and 0.9% sodium chloride. In this study, the determination of radiochemical purity of the one-system of TLC has been successfully developed using the Whatman-1 paper stationary phase and the mixture of mobile phase between acetone and 0.9% sodium chloride solution. The mobile phase of acetone: 0.9% sodium chloride with a ratio of 9:1 shows the most optimum results. This phase can separate 99mTc-DTPA (Rf = 0.4-0.6) from 99mTcO4- (Rf = 0.9-1.0) and 99mTcO2 (Rf = 0.0-0.1) as radiochemical impurities. This result shows that the one-system of TLC method can be used for radiochemical purity testing of 99mTc-DTPA radiopharmaceutical kits. This method can completely separate the product compound (99mTc-DTPA) from its impurities (99mTcO2 and 99mTcO4-).

Validation of TLC densitometry method for the quantitative determination of alkaloid in fermented endophytic fungi extract Phyllantus niruri Linn

Endophytic fungi can produce compound that similar with the host, so it can be used as mass production of compound. The optimal and valid assay method is needed to obtain the proper culture condition, which one using densitometric Thin Layer Chromatography (TLC). This research used 11 samples of endophytic fungi culture extract from Phyllantus niruri within various culture condition. Optimization of mobile phase was done using 3 kinds of mobile phase mixture, chloroform: ethyl acetate: methanol (8:8:4); chloroform: ethyl acetate: methanol: NH 4 OH (8:8:4:0.005); chloroform:ethyl acetate:NH 4 OH (8:8:0.005). Validation method measured using some parameters such as linearity, accuracy, and precision. The result of optimization and validation showed that TLC densitometry method can be used for measuring alkaloid level within the endophytic fungi extract of P. niruri using chloroform: ethyl acetate: NH 4 OH (8:8:0.005), with R value is 0.977, percentage of recovery is between 90–110, and RSD ≤7% on each concentration series. While the proper condition of endophytic fungi culture is using sucrose as carbon source and at pH 6 condition . The content of alkaoid reached 25.9 ± 1.4 mg 100 g -1 fresh weight.

Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid-liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 μm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5-80, 0.1-40 and 0.5-80 μg/mL) with average recoveries (100.64-108.28%, 98.48-105.91% and 97.68-101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients' follow-up especially in Egypt and other developing countries fighting HCV.

Development and validation of eco-friendly micellar-HPLC and HPTLC-densitometry methods for the simultaneous determination of paritaprevir, ritonavir and ombitasvir in pharmaceutical dosage forms

Ombitasvir, ritonavir and paritaprevir are three recently discovered directly acting antiviral drugs (DAADs) used in combined single dose tablet dosage form for treatment of hepatitis-C viral infections (HCV). The methods of analysis followed by quality control and research laboratories are required to be economic and fast; however, these methods can also produce huge amounts of chemical waste. In this study two fast, economic and green HPLC and HPTLC methods were validated for the simultaneous determination of the three drugs. For HPLC, isocratic elution used a mixture of micellar aqueous mobile phase consisting of (0.15 M sodium lauryl sulfate and 0.01 M sodium dihydrogen phosphate, pH 6.2) and ethanol (56:44). Elution was done on RP-C18 Kinetix® column (5 μm, 150 mm × 4.6 mm ID) at flow 1 mL min-1 and 254 nm UV-detector. HPTLC separations were performed on Merck® (20 cm × 10 cm) aluminum HPTLC plates coated with silica gel 60F254 using a mobile phase, Methylene chloride: methanol: ethyl acetate: ammonia (25%), (5:1:3:1, v/v/v/v) respectively. The calibration curves were linear across ranges of 3–100 μg mL-1 and 0.1–2 μg/spot for both HPLC and HPTLC methods, respectively. The two methods were applied successfully for the determination of the three drugs under study in their combined tablets dosage forms.

REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION AND FORCED DEGRADATION STUDIES OF EMTRICITABINE, RILPIVIRINE, AND TENOFOVIR ALAFENAMIDE IN SOLID DOSAGE FORM

Objective: A stability indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the estimation of emtricitabine (EMT), rilpivirine (RIL), and tenofovir alafenamide (TAF) in combined dosage forms and its API.Methods: Chromatographic separation was achieved on Waters ACQUITY RP-HPLC with PDA detector having Zodiac C18 Column (250×4.6×5μ) using mobile phase mixture of phosphate buffer: acetonitrile in the ratio of 40:60 v/v at 262 nm.Results: The assay was performed with tablets, and the % assay was found to be 100.104 for EMT, 99.74 for RIL, and 102.41 for TAF which shows that the method is useful for routine analysis. The linearity was found to be linear with a correlation coefficient of 0.999, which shows that the method is capable of producing good sensitivity. The retention time (RT) of EMT, RIL, and TAF using optimum conditions was found to be 2.517, 3.273, and 6.697 min. Forced degradation studies (FDS) were performed on sample using acid, base, thermal, photolytic, and peroxide degradation.Conclusion: Due to its simplicity, rapidness, high precision, and low RT value, this method was successfully applied to the estimation of EMT, RIL, and TAF combined dosage form. The drugs were found to be stable at FDS, and the net degradation was found to be within the limits.

REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION AND FORCED DEGRADATION STUDIES OF EMTRICITABINE, RILPIVIRINE, AND TENOFOVIR ALAFENAMIDE IN SOLID DOSAGE FORM

Objective: A stability indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the estimation of emtricitabine (EMT), rilpivirine (RIL), and tenofovir alafenamide (TAF) in combined dosage forms and its API.Methods: Chromatographic separation was achieved on Waters ACQUITY RP-HPLC with PDA detector having Zodiac C18 Column (250×4.6×5μ) using mobile phase mixture of phosphate buffer: acetonitrile in the ratio of 40:60 v/v at 262 nm.Results: The assay was performed with tablets, and the % assay was found to be 100.104 for EMT, 99.74 for RIL, and 102.41 for TAF which shows that the method is useful for routine analysis. The linearity was found to be linear with a correlation coefficient of 0.999, which shows that the method is capable of producing good sensitivity. The retention time (RT) of EMT, RIL, and TAF using optimum conditions was found to be 2.517, 3.273, and 6.697 min. Forced degradation studies (FDS) were performed on sample using acid, base, thermal, photolytic, and peroxide degradation.Conclusion: Due to its simplicity, rapidness, high precision, and low RT value, this method was successfully applied to the estimation of EMT, RIL, and TAF combined dosage form. The drugs were found to be stable at FDS, and the net degradation was found to be within the limits.

Stability indicating RP-UPLC method for the simultaneous determination of Atorvastatin and Amlodipine

Amlodipine is a calcium channel blocker. Atorvastatin is used for lowering the cholesterol. A new stability indicating liquid chromatographic method (RP-UPLC) has been proposed for the simultaneous estimation of Atorvastatin and Atorvastatin. Acquity UPLC HSS C18 column was used for the chromatographic study and a mobile phase mixture consisting of 0.1% Formic acid: Acetonitrile: Methanol (0.1:45: 55, v/v)/ 0.3/220 (Flow rate: 0.3 mL/min) (PDA detector 220 nm) was used for the elution of these two drugs. Stress degradation studies were performed and the method was validated as per ICH guidelines.

Stability indicating methods for selective determination of Oxetacaine in the presence of its degradation products

Two methods, HPLC and TLC were presented for the determination of Oxetacaine (OXT) in the existence of its different degradation products. HPLC method was based on the separation of OXT from its degradation products using reversed phase C18 column at room temperature and isocratic elution with mobile phase mixture of acetonitrile: 5 mM sodium dihydrogen orthophosphate dihydrate, pH was adjusted to 2.4 with orthophosphoric acid (50:50 v/v). Quantitation was based on peak area at 210 nm. The second TLC-densitometric method relies on the separation and quantitation of OXT from its degradation products on TLC silica gel 60 F254 plates, using 2-propanol: triethylamine (10:0.5 v/v) as a developing system and densitometric measurement of the developed bands at 210 nm. Validation of the proposed methods was performed according to the ICH guidelines and applied to evaluate the stability of OXT under different stress conditions.

Determination of Menaquinone-7 by a Simplified Reversed Phase- HPLC Method.

An efficient and accurate HPLC method was developed for the determination of menaquinone-7 (MK-7) in microbial fermentation using 2-propanol and n-hexane as extraction solvents as well as the eluent. Extraction was carried out with 2-propanol and n-hexane (2:1, v/v) after enzymatic hydrolysis with 1% (w/v) lipase and ethanol water treatment prior to quantification in order to remove interfering lipids and denatured proteins. Chromatographic separation of MK-7 was accomplished isocratically on a C 18 Gemini column using a mobile phase mixture of 2- propanol: n-hexane (2:1, v/v) with a flow rate of 0.5 mL/min. UV detection was carried out from 200-400 nm and the chromatogram was extracted at a wavelength of 248 nm. A linear response was shown by the method with a coefficient of determination (R2) value of 0.9982. The recoveries of MK-7 were greater than 94% and the intra and inter day R.S.D values were less than 2%, demonstrating the accuracy of the method. The lower limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/mL and 0.29 µg/mL, respectively. The general usefulness of the described method is demonstrated by the application of this method in the analysis of MK-7 from Bacillus species. Under these conditions, the analysis of MK-7 was achieved in less than 8 minutes with a retention time of 7.19 ± 0.1 minutes.

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.

Simultaneous Analysis of 6-Mercaptopurine, 6-Methylmercaptopurine, and 6-Thioguanosine-5’-monophosphate in Dried Blood Spot Using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

6-Mercaptopurine is a chemotherapeutic agent of the antimetabolite class. This study aims to analyze simultaneous validation of 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanosine-5’-monophosphate (6-TGMP) in dried blood spot (DBS) using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An accurate volume of 60 μL blood was spotted onto DBS-CAMAG paper and then extracted using methanol 90% (v/v) containing an internal standard of 5-fluorouracil (5-FU). Separation was performed using a Waters Acquity UPLC BEH AMIDA column 1.7 μm (2.1 x 100 mm) with a mobile phase mixture of 0.2% (v/v) formic acid in water−0.1% (v/v) formic acid in acetonitrile-methanol with gradient elution and flow rate of 0.2 mL/min. Mass detection was done using Waters Xevo TQD with positive electrospray ionization (ESI) for 6-MP, 6-MMP, 6-TGMP and negative ESI for 5-FU, in multiple reaction monitoring mode. Detection rates of 6-MP, 6-MMP, 6-TGMP and 5-FU were m/z 153.09 &gt; 119.09; 167.17 &gt; 126.03; 380.16 &gt; 168.00); 129.09 &gt; 42.05, respectively. This method is linear across the range 25.5–1020 ng/mL for 6-MP, 6-MMP and 6-TGMP. This method is valid for the in vitro simultaneous analysis of 6-MP, 6-MMP and 6-TGMP in DBS, based on European Medicine Agency guidelines.

Fast and sensitive method for the determination of trace 1,2,4-triazole and 4-amino-1,2,4-triazole by hydrophilic interaction liquid chromatography (HILIC) in antifungal drug substance

An HILIC method was developed to quantitatively determine trace amounts of 1,2,4-triazole and 4-amino-1,2,4-triazole in fluconazole drug substance. Separation was achieved on HILIC column using isocratic elution with mixture of water, acetonitrile and phosphoric acid mobile phase and UV detection at 205 nm. The method was validated according to ICH guideline requirements and was determined to be linear in the range of 0.6–22.0 μg/mL. Method accuracy and precision were assessed by spiking 1,2,4-triazole and 4-amino-1,2,4-triazole at three levels over the range of 2.5–15 μg/mL, the recoveries ranged from 78.6% to 95.7% for 1,2,4-triazole and 94.6% to 103.4% for 4-amino-1,2,4-triazole. The intermediate precision was represented by the overall %RSD of 6.9% (n = 9) for 1,2,4-triazole and 3.6% (n = 9) for 4-amino-1,2,4-triazole. The method was also shown to be repeatable at concentration levels of 2.5, 12.5 and 15 μg/mL with n = 3 %RSD values of 3.7%, 1.0% and 1.3% for 1,2,4-triazole, and 0.3%, 1.2% and 0.6% for 4-amino-1,2,4-triazole. The method was found to be specific, stability-indicating and has a detection limit of 0.8 μg/mL and a quantitation limit of 2.5 μg/mL. Finally, the method was demonstrated to be robust to small variations of key chromatographic parameters.

FACILE AND SENSITIVE HPLC-UV METHOD FOR DETERMINATION OF NINTEDANIB IN RAT PLASMA

Objective: In this study, a facile and sensitive high-performance liquid chromatographic method for determination of nintedanib in rat plasma was developed and validated.Methods: After plasma protein was precipitated by addition of acetonitrile, the supernatant underwent centrifugation. An aliquot was then injected into a high-performance liquid chromatographic system with a Mightysil RP-18 GP II ODS column (250 × 3.0 mm, length by inner diameter, 5-μm particle size) maintained at 50 °C. A mobile phase mixture of 20 mmol phosphate buffer (pH 3.0) and acetonitrile (7:3, v/v) was used at a flow rate of 0.6 mL/min, with UV detection at a wavelength of 390 nm for isocratic separation and detection of nintedanib, its main metabolite (BIBF1202), and p-nitrophenol as an internal standard.Results: The quantitative range of nintedanib concentration in this method was 12.5–400 ng/ml, and the calibration curves were linear. The intra-and inter-day accuracy values (relative errors) were in the range of −3.65%–4.00% and −3.65%–3.64%, respectively. The intra-and inter-day precision values (relative standard deviations) were&lt;5.9% and 8.36%, respectively. The method was successfully applied to a pharmacokinetic analysis of nintedanib in rats after intravenous administration.Conclusion: In this study, a rapid, sensitive, and simple HPLC-UV method for the quantitation of nintedanib in rat plasma was developed and validated. The method was shown to be accurate and precise and was successfully applied to a pharmacokinetic study.

HPLC – Quality by Design Approach for Simultaneous Detection of Torsemide, Spironolactone and Their Degradant Impurities

Abstract A simple, highly robust (quality by design (QbD) approach), precise and accurate method using high performance liquid chromatography coupled to mass spectrometry has been established for the simultaneous separation, identification and quantitation of a Torsemide (TOR), spironolactone (SPI) and their degradant impurities. The chromatographic separations of drugs and impurities were achieved on a inertsil ODS-3 µm C18, 150 mm × 4.6 mm, while the isocratic elution using a ternary mobile phase mixture of methanol, acetonitrile and water (5:3:2 v/v/v) at a flow rate of 0.2 mL/min was adopted for achieving optimum separations. The quantitation of torsemide and spironolactone was accomplished by UV detection at 254 nm and identification of the degradants were done by comparing identical mass in mass spectrometer. The recoveries of the torsemide and spironolactone were obtained higher than 98 % with good validation parameters; linearity (r2&gt;0.994), LOD and LOQ was 10 and 33 ng for TOR and 75 and 248 ng for SPI respectively. The quality by design (QbD) approach has been successfully utilized to prove the method is robust even deliberate changes in critical parameters.

Development of a stability-indicating UPLC method for determination of isotretinoin in bulk drug

A highly sensitive and rapid stability indicating ultra-performance liquid chromatographic (UPLC) method was developed for the quantification and identification of isotretinoin in bulk. Chromatographic separation was developed using a gradient elution in a reversed-phase system at flow rate of 0.5 ml/min with 12 min run time. The mobile phase was a gradient mixture of mobile phase A (contained a 30:70:0.5 mixture solution of methanol/purified water/glacial acetic acid) and mobile phase B (contained a 70:25:4.5:0.5 mixture solution of methanol/acetonitrile/purified water/glacial acetic acid). Eluents were monitored at 355 nm. The analytical method was validated for accuracy, precision, robustness, linearity, and forced degradation in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) topic Q2 (R1) ‘Validation of Analytical Procedures: Text and Methodology’. The method was linear over a concentration range of (1–7 µg/ml) with correlation coefficient of (r2 > 0.9999). The accuracy was confirmed by calculating the % recovery which was found to be 100.0–101.6%. The RSD values obtained for repeatability and intermediate precision experiments were less than 2%. The limit of detection (LOD) was 0.12 µg/ml, while the limit of quantification (LOQ) was 0.38 µg/ml. The drug samples were exposed to different stressed conditions and the results showed that all degradation products were satisfactorily separated from each other and from the peak of the drug using the developed method. The proposed method can be used for the quantitative determination of isotretinoin with confidence.