UPLC

The present research work describes comprehensive stress testing of eprosartan mesylate (EM) according to ICH guideline Q1A (R2), and development of a stability-indicating reversed phase ultra performance liquid chromatographic (UPLC) assay. The drug was ...

Objective: Aim of the present work is to develop a stability indicating ultra performance liquid
chromatography (UPLC) method to determine Lidocaine and its degradation impurities in pharmaceutical
dosage forms.
Method: Chromatographic separation was achieved by gradient elution on Agilent eclipse plus C18 (100x4.6)
mm, and 1.8μm column with potassium dihydrogen phosphate buffer (pH 4.50) and acetonitrile within a short
runtime of 14.0 min. The eluted compounds were monitored at wavelength of 230 nm using photodiode array
(PDA) detector, the flow rate was 1.0 mL/min, and the column oven temperature was maintained at 40 ◦C.
Result: The resolution of Lidocaine and six (potential, bi-products and degradation) impurities was greater
than 2.0 for all pairs of components. The repeatability and intermediate precision, expressed by the RSD, were
less than 1.0%. The accuracy and validity of the method were further ascertained by performing recovery
studies. The specificity of the method was investigated under different stress conditions including hydrolytic,
oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take
place under oxidative condition.
Conclusion: Method was Robustness against small modification in pH, column oven temperature, flow rate
and percentage of the mobile phase composition was ascertained. All these results provide that the method has
stability indicating properties being fit for its intended purpose; it may find application for the routine analysis
of the related substances of Lidocaine formulations.
Keywords: Ultra Performance Liquid chromatography (UPLC); Lidocaine; Validation; Stress Degradation
products

Quality-by-design-based UPLC method was developed for chromatographic separation to quantify
the antischizophrenic drug brexpiprazole in the presence of impurities. Research findings from pHscouting
studies were used as control variables which influence the chromatographic separation.
The peak tailing and resolution are the response variables and established the design-space by DoEstudy
for selection of suitable chromatographic conditions. Separation was achieved with lower
particle size stationary phase and buffer pH 2.0 in the mobile phase. The present method developed
through C18 50 × 2.1 mm, Ethylene-Bridged-Hybrid technology column with 1.7 μm particles,
mobile phase consists of pH 2.0 buffer and acetonitrile (67:33 v/v), flow rate of 0.5 mL min−1 and
detection wavelength at 215 nm. The retention time of brexpiprazole is 0.6 min and all impurities
were eluted within 2 min. The method linearity ranges were 20.4–61.3 μg mL−1 for assay and
0.88–6.59 μg mL−1 for dissolution with correlation-coefficients of 0.9999 and 0.9998 for assay and
dissolution, respectively. The recovery values were found in between 99.3 and 100.9%. The method
shows stability-indicating on the basis of noninterference of placebo, and impurities from forceddegradation
studies. Method validation was carried out according to ICH guideline Q2 (R1).

A methodical design-of-experiments were performed by applying quality-by-design concepts to establish
a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the
presence of degradation products. Response-surface, central-composite design, and quadratic model
were employed for statistical assessment of experimental data using the Design-Expert software.
Response variables such as resolution and retention time were analyzed statistically for chromatographic
screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were
considered for method optimization. The method was developed using C8 [100 � 2.1 mm, 1.8 μ] UPLC
column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50
v/v, the flow rate of 0.4 mL minute−1 and UV detection at 285 nm for both Carvedilol and Ivabradine.
The method was developed with a short run time of two minutes. The method was found to be linear in
the range of 25.0–199.9 μg mL−1 and 8.9–21.3 μg mL−1 for Carvedilol and Ivabradine, respectively with a
correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8%
and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH
Q2 (R1) guidelines.

A systematic design-of-experiments were performed by applying quality-by-design concepts to determine design space for rapid quantification of Teriflunomide by UPLC method in the presence of degradation products. Response surface and central composite quadratic were employed for statistical evaluation of experimental data using a Design Expert
software. The response variables such as resolution, retention time and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D and design space were studied. The method was developed through UPLC BEH C18 2.1x100mm, 1.7µ column, mobile phase comprised of buffer (5mM K2HPO4 containing 0.1% triethylamine, pH 6.8) and acetonitrile (40:60 v/v), the flow rate of 0.5 mL minute-1
and UV detection at 250nm. The method was developed with a short run time of one minute. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28-84 µg mL-1 , 2.8-22.7 µg mL-1 with a correlation coefficient
of 0.9999, 1.000 for assay and dissolution respectively. The recovery values were found in the range of 100.1-101.7%. The method was validated according to ICH guidelines.

In the present study total twelve impurities of Bisoprolol (BISO) and Hydrochlorothiazide
(HCTZ) were separated simultaneously in a single HPLC method. Out of these 12 impurities,
five are potential degradants, which are validated as per ICH guidelines. As the two active drug
substances BISO and HCTZ have different solubilities and polarities, the most critical
parameters in resolving the components from each other are pH, temperature, and solvents. The
method is precise (RSD<1.0%), accurate, linear (r2>0.999), robust, and stability indicating in the
range of LOQ to 150%. The HPLC method is then migrated to UPLC to further reduce the, run
time, solvent consumption, and increase the sample through put.

Degradation pathway for Rilpivirine Hydrochoride is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Rilpivirine Hydrochoride is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Six impurities are studied and the major degradant (RRT about 0.52) is identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5 %. Efficient chromatographic separation is achieved on a Shimpack XR –ODS-II stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 295 nm at a flow rate of 1.0 mL min-1.In the developed LC method the resolution between Rilpivirine Hydrochoride and six potential impurities (imp-1, imp-2, imp-3, imp-4, imp-5 and imp-6) is found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.995 for Rilpivirine Hydrochoride and six potential impurities. This method is capable to detect the impurities of Rilpivirine Hydrochoride at a level of 0.006% with respect to test concentration of 1.0 mg mL-1 for a 4-μL injection volume. The developed LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities and degradant determination.
Keywords: Rilpinavir Hydrochoride; UP-LC; LC-MS; Forced Degradation; Validation; Stability-Indicating.

A simple, précised and accurate RP-UPLC method has been developed and validated for the simultaneous assay of Telmisartan and Cilnidipine in tablets. Isocratic RP-UPLC method was developed on LC system of Waters Acquity UPLC with PDA detector on Water Acquity BEH C18, 2.1 x 100mm, 1.7μm column as stationary phase with binary gradient mode by using mobile phase as ACN: 0.01M sodium phosphates monobasic dehydrate buffer pH 3.0 with phosphoric acid (68:32, v/v), at a flow rate of 0.5 ml/min and the detection was carried out at 245 nm. Forced degradation study was carried out by oxidation, hydrolysis, photolysis and heating the drug. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the concentration range of 40-160 μg/ml with correlation coefficients of 0.9996 for Telmisartan and 10-40 μg/ml with correlation coefficients of 0.9995 for Cilnidipine. Degradation products produced as a result of stress studies did not interfere with the detection of Telmisartan and Cilnidipine: therefore, the assay can be considered to be stability-indicating.

In the present study total twelve impurities of Bisoprolol (BISO) and Hydrochlorothiazide
(HCTZ) were separated simultaneously in a single HPLC method. Out of these 12 impurities,
five are potential degradants, which are validated as per ICH guidelines. As the two active drug
substances BISO and HCTZ have different solubilities and polarities, the most critical
parameters in resolving the components from each other are pH, temperature, and solvents. The
method is precise (RSD<1.0%), accurate, linear (r2>0.999), robust, and stability indicating in the
range of LOQ to 150%. The HPLC method is then migrated to UPLC to further reduce the, run
time, solvent consumption, and increase the sample through put.

Natural red dyes have long been associated with power and extravagance, as they were expensive to acquire and the dyeing process was complex and required specialized knowledge. Until the end of the 15th century, cochineal, kermes and lac insects were collected and traded throughout Europe and Asia. At the beginning of the 16th century, the Castilians began exporting American cochineal from Mexico, which was richer in colorant than the European and Asian insects. This dyestuff became a great success in European and Asian centres of textile production, and a lucrative commodity for the economy of the Castilian Empire throughout the colonial period. This PhD project aims to take a closer look at the overall circulation of American cochineal as a commercial product and its dynamics in the process of revolutionizing ancient dyeing practices among 16th‐ and 18th‐century European and Asian textile workshops. ARCHLAB Access provided the opportunity to gain knowledge about the contents of th...

The present method provides the detailed description of development and validation of a simple stability indicating
liquid chromatographic method for Bupivacaine Assay in the presence of Methyl paraben. Successful separation of
the drug from methyl paraben was achieved with in 6min on C18, 50 x 4.6mm, 1.8μm column, using a 0.2M pH 6.8
Phosphate buffer, Acetonitrile and Milli Q water in the ratio of 5:40:650v/v/v at a flow rate of 1.0 mL per minute.
Column oven temperature was selected as 45ºC where the detection was carried out at 234 nm. The developed LC
method was validated with respect to specificity, linearity, accuracy, precision, ruggedness and robustness. The
assay method was found to be linear in the range of 25 μg·mL–1 to with 300 μg·mL–1 correlation coefficient of 0.9999
for methyl paraben and 125 μg·mL–1 to with 900 μg·mL–1 correlation coefficient of 0.9999 for Bupivacaine. A
recovery of assay of methyl paraben was found between 98.5% and 101.5% and Bupivacaine was found between
98.5% and 101.5%. The developed LC method to determine the assay of Bupivacaine can be used to evaluate the
quality of regular production samples and stability samples.

This study explores the ultra performance liquid chromatography for the quantification of mycophenolate mofetil (an immunosuppressant agent) in an injection formulation. Mycophenolate mofetil was separated from its seven specified impurities on HSS (high strength silica) C18 column (100 × 2.1 mm, 1.8 µm). The optimized mobile phase is compatible to mass spectrometry and hence the method can be equally run on LC-MS system for mass determinations. Efficient UV detection at 250 nm enabled determination of mycophenolate mofetil with no interference from placebo solution, diluent and other specified and unspecified impurities. The retention time of mycophenolate mofetil in the method was 1.79 min and all other impurities were eluted within in 8 min. The method optimization studies show that the selectivity of mycophenolate mofetil and its impurities was dependent on the type of organic solvent and its elution strength. The method was validated for specificity, linearity, precision, accuracy and robustness in range of 50 to 150 % of target analyte concentration (i.e. 62.50-195.31 µg mL⁻¹). The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. An excellent linear relation exists between the concentration and detector response (R² > 0.9999). Precision of the test method was proved with a relative standard deviation less than 1.0 %.

This study explores the ultra performance liquid chromatography for the quantification of Mycophenolate Mofetil (an immunosuppressant agent) in an injection formulation. Mycophenolate Mofetil was separated from its seven specified impurities on a HSS (High strength silica) C18 column (100 x 2.1 mm, 1.8 µm). The optimized mobile phase is compatible to mass spectrometry, and hence the method can equally run on LC-MS system for mass determinations. Efficient UV detection at 250 nm enabled determination of Mycophenolate Mofetil with no interference from placebo solution, diluent and other specified and unspecified impurities. The retention time of Mycophenolate Mofetil in the method was 1.79 minutes and all other impurities were eluted within in 6 minutes. The method optimization studies show that the selectivity of Mycophenolate Mofetil and its impurities was dependent on the type of organic solvent and its strength. The method was validated for specificity, linearity, precision, accuracy and robustness in range of 50 % to 150 % of target analyte concentration (i.e. 62.50 – 195.31 µg mL-1). The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. An excellent linear relation exists between the concentration and detector response (R2 > 0.9999). Precision of the test method was proved with a relative standard deviation less than 1.0%.

A simple, precise, and accurate RP-HPLC method has been developed and validated for the simultaneous assay of Telmisartan and Cilnidipine in tablets. Isocratic RP-HPLC method was developed on Waters C18250×4.6 mm, 5 μm column using mobile phase as acetonitrile (ACN): buffer pH 3.0 with orthophosphoric acid (68 : 32) at a flow rate of 1.0 mL/min and the detection was carried out at 245 nm using photodiode array detector. Forced degradation study was carried out by oxidation, hydrolysis, photolysis, and heating the drug. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was found to be linear in the concentration range of 40–160 μg/mL with correlation coefficient of 0.9990 for Telmisartan and 10–40 μg/mL with correlation coefficient of 0.9989 for Cilnidipine. Degradation products produced as a result of stress studies did not interfere with the detection of agomelatine; therefore, the assay can be considered to be ...

A precise, highly sensitive, selective and robust liquid chromatography-mass tandem spectrometry method was developed and validated for simultaneous quantification of Ticagrelor and Deshydroxyethoxy ticagrelor in human plasma by liquid-liquid extraction. Ticagrelor d-7 and Deshydroxyethoxy ticagrelor d-7 was used as an Internal standard. The reconstituted samples were analyzed on Eclipse XDB-C8 5μm 4.6*150mm column by using Acetonitrile and 0.1% Formic acid as the mobile phase in binary mode at the flow rate of 1.0 ml/min with a chromatographic run time of 3.0 min. The calibration curve developed was linear (r2 ≥ 0.99) in the concentration range of 2.5 to 1000 ng/mL for Ticagrelor and 1.0 to 300 ng/mL for Deshydroxyethoxy ticagrelor. All the analytes were found to be stable in various stability studies. The mean recovery for Ticagrelor, Deshydroxyethoxy Ticagrelor, Ticagrelor d-7 and Deshydroxyethoxy Ticagrelor d-7 were found to be 99.7%, 96.6%, 105.9% and 101.1% respectively. The limit of detection of the method was 0.5 ng/mL for Ticagrelor and 0.2 ng/mL for Deshydroxyethoxy ticagrelor. The developed and validated assay method was successfully applied to a pharmacokinetic study in human volunteers.

Context: Rubraxanthone has potential health benefits, such as antioxidant, anti-bacterial and cytotoxic agent. A sensitive method is required to quantify plasma concentrations to assess its efficacy.

Aims: To develop and validate an analytical method for the determination rubraxanthone in human plasma using ultra-performance liquid chromatography (UPLC-UV) for pharmacokinetic application.

Methods: Chromatographic separation was performed aced using a C18 column (100 mm × 3.0 mm, particle size 1.8 μm) with a mobile phase consisting of acetonitrile – 0.4% formic acid (75:25, v/v). The isocratic flow rate was 0.3 mL/min with elution time for rubraxanthone was approximately 3 min and UV detection were at 243 nm. Biological sample preparation involved protein precipitation method with acetonitrile. The developed method was validated as EMEA guidelines for its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability.

Results: The method was proven to be linear over a plasma rubraxanthone concentration range of 206 to 6180 ng/mL with a mean correlation coefficient of 0.999. The within-run and between-run precision (coefficient of variation) were less than 4.7%. The mean recovery of rubraxanthone from human plasma was found to be greater than 95%. The lower limits of quantitation of the method was determined to be 206 ng/mL. The samples remained stable during the processing and analysis times and also during the three freeze/thaw cycles.

Conclusions: The UPLC-UV method was validated for all of the criteria that were necessary for a bioanalytical method and could reliably quantitate rubraxanthone in human plasma for future clinical pharmacokinetic study.

Plerixafor (PLX) injections are administered to patients with cancers of lym-phocytes (non-Hodgkin's lymphoma) and plasma cells (multiple myeloma). The main objective of the current study was to develop a short reverse phase chromatographic method for the simultaneous quantification of PLX and its impurities, in an injection formulation, to reduce the time required for these quality tests. Furthermore, the present work describes the role of nonalkyl branched nonquaternary ion pair reagent in improving the peak shape and reducing column equilibration time. The separation of PLX and its related substances is pH dependent (optimum pH = 2.50) and was achieved on an octadecylsilyl (C18) column. The method was validated for its intended purpose in accordance with the current regulatory guidelines for validation. The proposed method can be applied for quality control, release, and stability analyses of active pharmaceutical ingredient, PLX, as well as finished products, PLX injections.

Epidemiological studies have linked whole grain consumption to prevention of several chronic diseases. Whole grain is a source of important phytochemicals, such as ferulic acid (FA). FA is the most abundant phenolic and major contributor to the in vitro antioxidant capacity of wheat grain. Several studies have reported highly variable results on FA bioavailability (0.4–98%). The binding of FA to polysaccharides may limit its bioavailability. Therefore, our study aimed at monitoring release features of FA during gastrointestinal (GI) transit. This was termed bioaccessibility. The bioaccessibility of FA was studied from different wheat fractions and breads with the use of a dynamic in vitro system that simulates the upper GI transit and digestion. The results showed low bioaccessibility of FA from the wheat fractions and breads (<1%). However, the bioaccessibility was high when free FA was added to flour (∼60%). The bioaccessibility of FA appeared to be determined by the percentage of free FA. In wheat grain, most of FA is bound to arabinoxylans and other indigestible polysaccharides restricting its release in the small intestine. New processing developments should be considered to increase free FA in the cereal matrix in order to improve its bioavailability and systemic health effect.