Ultra Performance Liquid Chromatography Technique Research Articles

A NEW RP-UPLC METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF DORZOLAMIDE HCl AND TIMOLOL MALEATE

Objective: This investigation demonstrates a stability-indicating and reliable “reverse-phase ultra-performance liquid chromatography” method to simultaneously quantify timolol maleate and dorzolamide HCl in the pharmaceutical dosage form. Methods: Successful separation was accomplished using Phenyl column (100 mm x 2.1 mm, 1.7μm) with isocratic type of elution using mobile phase containing Acetonitrile+Ammonium Formate buffer (30:70), respectively with 0.2 ml/min flow rate. The wavelength sensor was attuned at 266 nm to quantify timolol maleate and dorzolamide HCl. Results: Dorzolamide HCl and timolol maleate peaks were eluted with fine resolution at retention times 0.7 min and 1.5 min, respectively. In the 55.75-334.5 μg/ml and 6.25-37.5 μg/ml concentration ranges for dorzolamide HCl and timolol maleate, the calibration graphs were linear, with regression coefficients of 0.99997 and 0.99991, respectively. The suggested ultra-performance liquid chromatography approach has been shown as sensitive, precise, robust, accurate, specific and stability, indicating through the resolution of dorzolamide HCl and timolol maleate from its degradation-based compounds. Conclusion: The established ultra-performance liquid chromatography technique was effectively extended to the evaluation of dorzolamide HCl and timolol Maleate in the pharmaceutical dosage form and the test results appeared satisfactory.

Development of a Stability Indicating UPLC Method for the Determination of Tirbanibulin in Bulk and Its Pharmaceutical Dosage Form.

The primary goal of this study was to create and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for estimating tirbanibulin in pure and dosage form. A UPLC technique was developed using a Waters Acquity UPLC Phenyl (100 x 2.1 mm, 1.7 µm) column. The developed technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Tirbanibulin was separated chromatographically with high resolution using the mobile phase acetonitrile: buffer (30:70 v/v) at 0.5 mL/min, 5 µL injection volume, and 220 nm wavelength. The validated technique was found to be linear in the 1-15 µg/mL range. The detection and quantification limits for tirbanibulin were 0.03 and 0.1 µg/mL, respectively. The percentage relative standard deviation was less than 2%, demonstrating the precision of the developed technique. Furthermore, the recovery rate was nearly 100%, confirming the accuracy of the method. Minor modifications to the chromatographic conditions demonstrated the robustness of the method. The developed analytical method was precise, simple, reproducible, and sensitive. Consequently, it can be used to determine tirbanibulin.

Box–Behenken-Supported Development and Validation of UPLC Method for the Estimation of Eugenol in Syzygium aromaticum, Cinnamomum tamala, and Myristica fragrance

Eugenol (EUG) is one of the most important components available in several spices, including clove, bay leaves, and nutmeg. These spices are used as flavouring agents in foods and beverages. The aim of the present study is to develop and validate a rapid, simple, sensitive, and robust ultra-performance liquid chromatography (UPLC) technique for the quantitative estimation of EUG in the ultrasound-assisted methanolic extracts of three spices, namely Syzygium aromaticum (L.) Merr. & L.M.Perry (SA), Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm (CT), and Myristica fragrance Houtt. (MF). EUG was isocratically separated on a UPLC C18 column. The acetonitrile:methanol:water (50:40:10, v/v/v) solvent in different proportions was optimized as the mobile phase for the determination of EUG in ultrasound-assisted methanolic extracts of three different spices. The quantitative estimation of EUG was performed at a 281 nm detection wavelength. The column oven temperature was maintained at 35 ± 5 °C, and the flow rate of the mobile phase was 0.2 mL/min using an injection volume of 1 µL. The UPLC technique was validated according to the ICH guidelines and showed an excellent linearity range of 10–100 ng/mL. The robustness of the method was validated using Box–Behenken response surface design (BBD) software, and a 0.2 mL/min flow rate of the mobile phase, a column oven temperature of 308 K, and a 281 nm detection wavelength were found to be the best optimal conditions for obtaining the highest amount and separation of EUG. The content of EUG in ultrasound-assisted methanolic extracts of SA, CT, and MF using the UPLC technique showed 313.67 ± 0.87 mg g−1, 44.95 ± 0.56 mg g−1, and 59.66 ± 0.41 mg g−1, respectively. The antioxidant potentials of EUG, SA, CT, and MF were analysed using the DPPH (2,2-diphenyl-1-pcrylhydrazil radical) method, which revealed the antioxidant potential of EUG (IC50 = 3.12 µg/mL), standard ascorbic acid (IC50 = 7.06 µg/mL), SA ultrasound-assisted methanolic extract (IC50 = 5.97 µg/mL), CT ultrasound-assisted methanolic extract (IC50 = 49.48 µg/mL), and MF ultrasound-assisted methanolic extract (IC50 = 65.16 µg/mL). The proposed UPLC technique can be used to quantitatively assess various spices, plants, pharmaceutical products, and polyherbal formulations containing EUG as an active constituent.

A Validated Ultra Performance Liquid Chromatography Method for Simultaneous Estimation of Diacerein and Aceclofenac in Bulk and Pharmaceutical Formulation

A simple, exceptionally cost-effective, extremely accurate, quite precise and highly reproducible analytical method was developed and validated for simultaneous estimation of Diacerein and Aceclofenac in bulk and Pharmaceutical formulation. The new Ultra Performance Liquid Chromatography technique was developed. Method- The separation has been done on column ACQUITY UPLC BEH Shield C18 (50 x 2.1mm), 1.7µm (40°C temperature). The mobile phase contained Buffer and Acetonitrile (Buffer: ACN) (55:45 V/V).. The flow rate was set at 0.4ml/min, and detected at 268nm with PDA detector. Mobile phase was sonicated for 15 min. before use. 5μl of samples of standard stock solution and tablet solution were injected. Different trials were performed to separate diacerein and aceclofenac. The total run time of the detection was 4 min. The developed method was validated against different validation parameters. Results - The retention times were obtained at 1.762min. and 2.891 min. for Diacerein and Aceclofenac respectively. Accuracy study was observed in the range of 100.33% to 101.67% with less than 2% RSD. LOQ was found to be 2.98μg/ml and 5.9041μg/ml. similarly LOD was found to be 0.9841μg/ml and 1.9467μg/ml. for Diacerein and Aceclofenac respectively. Precision %RSD for intraday and interday were found to be 0.19 and 0.18 for Diacerein , 0.44 and 0.16 for Aceclofenac respectively. Linearity was found in the range of 2.5-17.5μg/ml and 5-35μg/ml for Diacerein and Aceclofenac respectively. The method was found robust by deliberate changes in the flow rate, ratio of mobile phase and detection wavelength. Conclusion-The method was found to be satisfactory and can be used successfully for determination of Diacerein and Aceclofenac simultaneously in bulk and pharmaceutical dosage form.

A NEW NP-UPLC METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF RAMUCIRUMAB AND ERLOTINIB

Objective: This investigation demonstrates a stability-indicating and reliable “normal phase ultra-performance liquid chromatography” method to simultaneously quantify Ramucirumab and Erlotinib in the pharmaceutical dosage form. Methods: Successful separation was accomplished using Chiralcel-OD-3 column (50 mm x 4.6 mm, 3 μm) with an isocratic type of elution using a mobile phase containing n-hexane+isopropyl alcohol+methanol (89:10:1), respectively with 1.0 ml/min flow rate. The wavelength sensor was attuned at 266 nm to quantify Ramucirumab and Erlotinib. Results: Erlotinib and Ramucirumab peaks were eluted with fine resolution at retention times 1.7807 min and 3.175 min, respectively. In the 10-150 μg/ml and 1-15 μg/ml concentration ranges for Erlotinib and Ramucirumab, the calibration graphs were linear, with regression coefficients of 0.99928 and 0.99976, respectively. The suggested ultra-performance liquid chromatography approach has been shown as sensitive, precise, robust, accurate, specific and stability indicating through the resolution of Erlotinib and Ramucirumab from its degradation-based compounds. Conclusion: The established ultra-performance liquid chromatography technique was effectively extended to the evaluation of Erlotinib and Ramucirumab in the pharmaceutical dosage form and the test results appeared satisfactory.

Development and Validation of Novel Ultra Performance Liquid Chromatography Technique for the Simultaneous Determination of Metformin and Repaglinide in Bulk and Formulation

A novel ultra-performance liquid chromatographic technique for the estimation of metformin and repaglinide in a API and tablet dosage form. The chromatographic separation was achieved using DIKMA Endoversil (2.1 x 50mm, 1.7µm) column with a mobile phase of phosphate buffer, pH 4.2 and methanol as a mobile phase (38:62) with a flow rate of 0.3 mL/min and the detection wavelength was monitored at 241 nm. The method was validated in accordance with International conference on harmonization guidelines. In this present method metformin was eleued at 0.516 minute and repaglinide was eluted at 1.152 min. Limit of detection was 0.05 μg/ml for metformin and1.152 μg/ml for repaglinide limit of quantification was found 0.5 μg/mL. Calibration curve plots were found linear over the concentration ranges 1-50 μg/mL for both the analytes. The % assay of the marketed dosage form was found 99.45 % for metformin and 97.08 % for repaglinide. The present study approach was found to be effective in the analysis of both analytes in force degradation conditions, because both the analytes has been specifically eluted in presence of other chromatograms. The experiential evidences of all the study results revealed the suitability of the estimation of metformin and repaglinide in API and tablet dosage form.

Method development and validation of Ifetroban by RP-UPLC

The purpose of this work is to develop and validate reverse phase Ultra performance liquid chromatography (UPLC) method for the rapid and precise determination of ifetroban sodium in its pure form and in formulations. A simple, specific, accurate, precise isocratic UPLC method for analysis of Ifetroban sodium was developed and validated using a Phenomenex C18 column (50 mm x 3.0 mm, 3µ) as the stationary phase, in conjunction using Triethyl amine buffer: methanol in the proportion of 25:75 with a flow rate of 1.0 mL/min, run time is 3 min and UV detector is used at 235 nm wavelength. The developed UPLC technique was found to be rapid as the retention time was 0.56 minutes for Ifetroban peak to elute. The developed UPLC technique was validated as per the ICH guidelines for specificity, linearity, accuracy, precision, robustness and found to be satisfactory. Linearity was established in the concentration range 100-300 µg/mL with correlation coefficient of 0.998 and the equation obtained is y = 0.635x + 0.639.The percentage recovery is 100.41. The method is rugged and is trouble free and transferable. The study showed that the developed UPLC technique can be used for the estimation of drug purity, stability, solubility and with no interference of pharmaceutical excipients from the active pharmaceutical ingredient. The precision, accuracy, robustness results obtained enables rapid quantification of ifetroban for quantitative analysis.

Stability-Indicating RP-UPLC Method for Simultaneous Determination of Azilsartan Medoxomil and Chlorthalidone in Tablets in the Presence of Its Degradation Products.

Azilsartan Medoxomil (AZL) angiotensin II receptor blocker and chlorthalidone (CLT) were determined by ultraperformance liquid chromatography (UPLC) method in their combined dosage form, they were both subjected to forced degradation studies under extensive stress conditions. The method is a stability indicating by resolving the investigated drugs from their degradation products. Moreover, the degradation products for both drugs obtained from forced degradation were subjected to LC-MS for structure elucidation. The UPLC technique depends on the measurement of spectra for AZL and CLT at 254 nm. Linearity, accuracy and intermediate precision were acceptable over the concentration range of 67.2-268.8 and 40-160 μg/mL for AZL and CLT, respectively. The method was applied for the determination of the studied drugs in their dosage forms. The UPLC method is inexpensive, simple and considered as green chemistry method for the routine analysis and quality control of both drugs in their combined dosage form.

Blue lupine seeds protein content and amino acids composition

Lupine seeds are promising soy replacement in food products and feeds. Eighteen cultivars of blue lupine seeds were examined to indicate the one most favourable in protein content and quality. Two parameters were studied, i.e. total protein content (with the Kjeldahl method) and amino acids composition (with the ultra performance liquid chromatography technique). Both parameters studied were variable and depended both on the cultivar and on the place of cultivation. Protein content was in the range of 28–41% and the worst cultivar, regardless of cultivation place, was cv. Kalif (average total protein content 29.37 ± 1.14%), while the best cultivar was Boruta (average total protein content 37.43 ± 0.98%). The blue lupine seeds were rich especially in leucine (5.3 ± 0.5–9.7 ± 0.5 g/16 g N), threonine (2.4 ± 0.7–4.9 ± 0.1 g/16 g N) and lysine (2.7 ± 0.4–5.6 ± 0.1 g/16 g N). The richest among all amino acids studied were the cv. Bojar seeds from Wiatrowo and cv. Oskar from Przebędowo. It was not possible to choose one cultivar preferable from the nutritional point of view, because a strong influence of cultivation place on protein content and quality was observed, especially as to the precipitation sum.

Solubility, thermodynamic properties and solute-solvent molecular interactions of luteolin in various pure solvents

The objective of this study was to evaluate solubility, thermodynamic properties and solute-solvent molecular interactions of poorly water-soluble bioactive compound luteolin (LUT) in various pure solvents namely water (H2O), ethylene glycol (EG), propylene glycol (PG), polyethylene glycol-400 (PEG-400), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), 1-butanol (1-BuOH), 2-butanol (2-BuOH), ethyl acetate (EA), dimethyl sulfoxide (DMSO) and Transcutol® (THP) at temperatures “T = 298.2 K to 318.2 K” and pressure “p = 0.1 MPa”. The solubility values of LUT at equilibrium were determined using a shake flask technique with the help of ultra-performance liquid chromatography technique at 348 nm. The solid state of LUT in pure and equilibrated form was characterized using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) techniques. The solubilities of LUT measured by shake flask method were correlated with “van't Hoff and Apelblat models”. The DSC and PXRD spectra of pure and equilibrated LUT were similar, suggesting no transformation of LUT during equilibrium. Good correlation was recorded between experimental and calculated solubilities of LUT. The solubility values of LUT (mole fraction) were obtained maximum in PEG-400 (1.27 × 10−1) followed by DMSO (7.15 × 10−2), THP (4.01 × 10−2), 2-BuOH (5.53 × 10−3), 1-BuOH (5.36 × 10−3), EA (5.11 × 10−3), IPA (4.14 × 10−3), PG (3.89 × 10−3), EtOH (3.08 × 10−3), EG (2.38 × 10−3), MeOH (8.16 × 10−4) and H2O (4.17 × 10−6) at “T = 318.2 K” and similar trend was obtained at each temperature studied. The physical values of activity coefficients were determined with the help of ideal solubilities of LUT. Based on the physical values of activity coefficients, maximum solute-solvent interactions were observed in LUT-PEG-400, LUT-DMSO and LUT-THP. Apparent thermodynamic analysis suggested endothermic and entropy-driven dissolution of LUT in each pure solvent studied.

Graphite-Based Nanocomposite Electrochemical Sensor for Multiplex Detection of Adenine, Guanine, Thymine, and Cytosine: A Biomedical Prospect for Studying DNA Damage.

Guanine (G), adenine (A), thymine (T), and cytosine (C) are the four basic constituents of DNA. Studies on DNA composition have focused especially on DNA damage and genotoxicity. However, the development of a rapid, simple, and multiplex method for the simultaneous measurement of the four DNA bases remains a challenge. In this study, we describe a graphite-based nanocomposite electrode (Au-rGO/MWCNT/graphite) that uses a simple electro-co-deposition approach. We successfully applied the developed sensor for multiplex detection of G, A, T, and C, using square-wave voltammetry. The sensor was tested using real animal and plant DNA samples in which the hydrolysis of T and C could be achieved with 8 mol L-1 of acid. The electrochemical sensor exhibited excellent sensitivity (G = 178.8 nA/μg mL-1, A = 92.9 nA/μg mL-1, T = 1.4 nA/μg mL-1, and C = 15.1 9 nA/μg mL-1), low limit of detection (G, A = 0.5 μg mL-1; T, C = 1.0 μg mL-1), and high selectivity in the presence of common interfering factors from biological matrixes. The reliability of the established method was assessed by method validation and comparison with the ultraperformance liquid chromatography technique, and a correlation of 103.7% was achieved.

Quantification of Valerenic Acid in Ferula sumbul Roots Using UPLC

Ferula sumbul (Umbelliferae) roots have a characteristic musk odour and it has been traditionally used for relieving anxiety, as a sedative in hysteria and other nervous disorders. Present investigation aims to develop and validate a simple, robust, accurate and fast analytical ultra-performance liquid chromatography technique for the estimation of valerenic acid in F. sumbul roots. F. sumbul root extract was prepared by refluxing the powdered roots with methanol for 30 minutes. A simple method for the estimation of valerenic acid has been developed and validated using ultra-performance liquid chromatography with photodiode array detector. The estimation was achieved using BEH Shield RP, C-18 column (2.1×50 mm, 1.7 μm) with isocratic elution of methanol and 0.5% phosphoric acid (75:25) at a flow rate of 0.25 ml/min and detection at 218 nm. The method has been developed for the first time and is accurate with good linearity (r2>0.998). F. sumbul roots were found to contain 12.62±0.074 µg/g valerenic acid. The developed method can be selectively used for the estimation of valerenic acid in the roots of F. sumbul. All the validation parameters were found to be within the permissible limits prescribed by ICH guidelines.

Antimutagenic effects of aqueous fraction of Myristica fragrans (Houtt.) leaves on Salmonella typhimurium and Mus musculus.

Natural plant extracts offer a promising hope in the prevention/treatment of cancer arising from genetic mutations. This study evaluated in vitro and in vivo mutagenic and antimutagenic effects of aqueous fraction of Myristica fragrans (AFMF) leaves on TA100 strain of Salmonella typhimurium and Mus musculus (Male Swiss albino mice), respectively. The antioxidant activity of AFMF against 2,2-diphenyl-1-picrylhydrazyl (DPPH), total phenolic and flavonoid contents were determined, followed by its phytochemical elucidation using the Ultra Performance Liquid Chromatography technique (UPLC). The mutagenicity of AFMF at 4, 20, 50, 100, 200, 500, and 1000 µg/well was <2.0 in S. typhimurium and the induced micronucleated polychromatic and normochromatic erythrocytes at 500, 1000, 2000, and 4000 mg/kg were not significantly different from the negative control (p≥0.05). The mutagenic activity of benzo[a]pyrene and cyclophosphamide was significantly suppressed above 50.0% throughout the tested concentrations. Fifty percent of the free radicals from DPPH were scavenged by AFMF at 0.11 mg/ml. Total phenolic and flavonoid contents of AFMF were 51.0 mg GAE/g and 27 mg QE/g, respectively. Rutin was elucidated by the UPLC technique, and thereby suspected to be the phytochemical responsible for the observed antimutagenic activity. Thus far, AFMF seems to contain a promising chemotherapeutic agent for the prevention of genetic damage that is crucial for cancer development.

Screening nephroprotective compounds from cortex Moutan by mesangial cell extraction and UPLC.

A method for screening nephroprotective compounds in cortex Moutan, a common traditional Chinese medicine (TCM) in treating diabetic nephropathy with renal mesangial cell extraction and ultra performance liquid chromatography technique was described in this paper. We hypothesize that the compounds which bind to cell membranes under pathological conditions may be the bioactive compounds in TCMs. Mesangial cells were cultured in medium containing 5 mM (physiological, NG) or 30 mM (pathological, HG) glucose for 48 h and then incubated with cortex Moutan extract. After the unbound substances were washed off, the cell membrane-bound compounds were dissociated and concentrated by an SPE column. By comparing the chromatograms of NG and HG cultured-cell extractions and cortex Moutan extract, three compounds bound to both NG and HG-cultured mesangial cells were identified as paeoniflorin, pentagalloylglucose (PGG) and paeonol. In vitro studies showed that paeoniflorin, PGG and paeonol reduced the activity of nicotinamide-adenine dinucleotide phosphate oxidase (NADPH) activity, and decreased the level of reactive oxygen species (ROS), transforming growth factor-β1 (TGF-β1) and fibronectin in high glucose cultured mesangial cells. The results indicate that paeonol, paeoniflorin and PGG may be the nephroprotective compounds from cortex Moutan. This study is expected to provide a more reliable and effective method for screening bioactive compounds from the complex TCM systems.

Development of a stable isotope dilution UPLC‐MS/MS method for quantification of dexmedetomidine in a small amount of human plasma

Dexmedetomidine (Dex) is a selective central α2-agonist with anesthetic properties and has been used in clinical practice for sedation in the intensive care unit (ICU) after operations. In this study, an analytical assay for the determination of Dex in a small amount of plasma was developed for the application to pediatric ICU trials. The quantification of Dex was constructed using the original stable isotope Dex-d3 for electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in the selected reaction monitoring mode. A rapid ultra-performance liquid chromatography technique was adopted using ESI-MS/MS with a runtime of 3 min. Efficacious concentration levels (50 pg/mL to 5 ng/mL) could be evaluated using a very small amount of plasma (10 μL) from patients. The lower limit of the quantification was 5 pg/mL in the plasma (100 µL). For sample preparation, a solid-phase extraction was used along with the OASIS-HLB cartridge type. Recovery values ranged from 98.8 to 100.3% for the intra- [relative standard deviation (RSD), 0.9-1.3%] and inter- (RSD, 0.9-1.5%) day assays. A stable test had recovery values that ranged from 97.8 to 99.7% with an RSD of 1.0-1.9% for the process/wet extract, bench-top, freeze-thaw and long-term tests. This method was used to measure the Dex levels in plasma from pediatric ICU patients. In the clinical ICU trial, the small amount of blood (approximate plasma volume, 200 μL) remaining from blood gas analysis was reused and targeted for the clinical analysis of Dex in plasma.

Development of an Ultra-performance Liquid Chromatography Technique Coupled with Mass Spectrometry for the Measurement of Tacrolimus in Micro-samples of Whole Blood, and its Application on a Pharmacokinetic Trial

The aim was to develop a rapid, specific, sensitive and accurate chromatographic technique coupled with mass spectrometry for the measurement of tacrolimus (CAS 104987-11-3) in microsamples of whole blood, and its application on a pharmacokinetic pilot trial. A fast gradient was designed in an ultra-performance liquid chromatography, and coupled with a mass spectrometer for the quantification of tacrolimus in 100 microl samples of EDTA whole blood. Multiple reaction monitoring was used for the measurement of tacrolimus (m/z(+1) 821.49-->4768.35 Th) and sirolimus as internal standard (m/z(+1) 931.69-->864.39 Th). The method was validated according to Mexican regulatory guidelines. Twenty-four young healthy male volunteers with similar hematocrit values participated in the pharmacokinetic trial; an oral single dose of one 5 mg tacrolimus capsule was administered and kinetic profiles were described since 0 h until 24 h post-dose. Method showed to be accurate, precise and linear over the range from 1 to 80 ng/ml, having an absolute recovery of 94%. Molecule was stable for two months at -70 degrees C, and heparin interfered with its quantification. Total run-time is around 1.5 min. Mean maximum blood concentration was 32.63 +/- 1.74 ng/ml, and was reached at 1 h post-dose; elimination half-life was 14.18 +/- 5.71 h. Method developed is not time-consuming, inexpensive, and sensitive enough for its application during pharmacokinetic trials, and can be suitable for therapeutic drug monitoring in transplanted patients. Pharmacokinetic data obtained in Mexican population are quite similar to previously reported in international literature.