Rapid Reversed-phase HPLC Method Research Articles

Development of a high-performance liquid chromatography method for rapid radiochemical purity measurement of [18 F]PSMA-1007, a PET radiopharmaceutical for detection of prostate cancer.

Since first becoming commercially available in 2018, the PET radiopharmaceutical [18 F]PSMA-1007 has been used widely for the diagnosis and staging of prostate cancer. A pharmacopoeia monograph first became available in 2021, prescribing a radiochemical purity specification of >91%, based on analytical results from both TLC (for [18 F]fluoride impurity alone) and HPLC (for all other 18 F-impurities). Though this monograph has provided clarity for the quality control testing of [18 F]PSMA-1007, it prescribes a HPLC method using phosphate buffer mobile phase that may present a risk of precipitation of phosphate salts in the HPLC system. The method also requires specialised hardware not immediately available to all laboratories. This work describes the development of a simple, rapid reversed-phase HPLC method utilising 0.1 M ammonium formate mobile phase for the accurate assessment of both [18 F]fluoride impurity and overall radiochemical purity in a single test. This method is especially useful for assessment of product stability over time. A more accurate TLC method for [18 F]fluoride impurity is also described.

Validated Reverse Phase-High-Performance Liquid Chromatography Method for Simultaneous Determination of Vanillic acid and Glyburide in Amphiphilic Polymeric Micelles

Background: A new sensitive and rapid reverse phase HPLC method was developed for the simultaneous determination of vanillic acid (VA) and glyburide in the formulation and validated according to (ICH) Q2 (R1) guidelines. Methods: The HPLC analysis was performed using the C-18 reverse phase column and a mobile phase consisting of acetonitrile and orthophosphoric acid (0.1% v/v) of ratio 70:30 v/v at 1 mL/min of flow rate. The detection was performed at the wavelength (λ) of 234 nm (isosbestic point), and the retention time of VA and glyburide was found around 2.6 and 5.4 min. The calibration plot gave a linear relationship over the concentration range of 2–10 μg/mL with a regression coefficient of 0.999. The LOD and LOQ for VA were 0.34 and 1.04 μg/mL, while for glyburide it was 0.38 μg/mL and 1.17 μg/mL respectively. The accuracy of the proposed method was determined by recovery studies and was in the range of 95% to 105%. The RSD% of the determination of precision was <2%. Results: The results of the robustness study were within the acceptable limits in response to changes in flow rate, ratio of mobile phase, and pH. The method was successfully applied for the determination of EE% and drug release from amphiphilic polymeric micelles. Conclusion: The EE% of both the drugs prepared in a liquid formulation of amphiphilic polymeric micelles was found to be greater than 90%. The results of drug release studies indicated almost 100% VA and 85.3% glyburide release using pH-gradient method within 4 and 48 h respectively.

Rapid reversed-phase high performance liquid chromatography assay of Tert-butylhydroquinone content in food products

The use of antioxidant has been widely applied in food industry to maintain the product’s condition to the end consumer. Tert-butylhydroquinone (TBHQ) is a synthetic aromatic organic compound which is often used as antioxidant in food, especially oil product and its derivatives. The use of TBHQ in food products has been limited by the government in most countries, including Indonesia. Therefore, the need for a rapid method to assay the concentration of TBHQ in products is quite high in food industry. In this article, we propose a rapid reversed-phase HPLC method to assay TBHQ content in food products. The isocratic system using 1 % acetic acid and acetonitrile in 3:2 ratio with 1 mL min−1 flow rate produces good separation of TBHQ. Target peak was eluted in 5 min and the whole procedure only need maximum of 18 min to elute all the sample component. The LOD of the method was determined at 0.2 mg L−1, while the lower limit of quantification was 0.5 mg L−1. The accuracy of the method has been proved with acceptable recovery ranging from 95 % to 105 %.

Development of Stability Indicating and Robust RP-HPLC Method for Determination of Teneligliptin

A simple and rapid reverse-phase HPLC method was developed for determination of Teneligliptin (TGP) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 ACE column (150x 4.6 mm i.d.; 5 μm) using mobile phase as a mixture of Phosphate buffer pH-7.2 using ortho-phosphoric acid: methanol (30:70v/v). The UV detection was carried out at 245nm at ambient temperature and the flow rate of 1.0 mL/min. The calibration curve was found to be linear in the concentration range of 10-50 μg/mL(r=0.9993). Force degradation study was performed under various conditions like acidic, alkaline, oxidative, photolytic and mass balance calculations were carried out from the degradation results. The developed method was validated as per ICH guidelines with respect to linearity, accuracy, precision, limit of detection and quantification. The robustness of the proposed method was evaluated by the Plackett Burman design. The purity of the degraded sample was checked by peak purity analysis. The peaks of degradation products did not interfere with that of pure Teneligliptin.

Stability Study of Etoricoxib a Selective Cyclooxygenase-2 Inhibitor by a New Single and Rapid Reversed Phase HPLC Method

Cyclooxygenase-2 (COX-2) is an enzyme responsible for inflammation and pain. Etoricoxib is the most recent selective (COX-2) inhibitor that has a higher COX-2 selectivity than the other COX-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs), which significantly improves its gastric safety profile. The current therapeutic indications of etoricoxib includes the treatment of several painful conditions, such as osteoarthritis, acute gout, ankylosing spondylitis, and rheumatoid arthritis. To the best of found knowledge, no decent method has been reported that can be used for the routine determination of etoricoxib and additives in pharmaceutical suspensions by a single, rapid and cost-effective run of HPLC, using an UV-Vis detector. Earlier reported methods, such as liquid chromatography-mass spectrometry (LC-MS), high performance thin layer chromatography (HPTLC), capillary zone electrophoresis, and ultra performance liquid chromatography (UPLC), are all tedious and time consuming. A reversed phase high performance liquid chromatography (RP-HPLC) was used as a first reported single run method to achieve developed and validated simultaneous determination for sodium saccharin, vanillin, methyl paraben, etoricoxib, and butyl paraben, in prepared oral suspensions of etoricoxib. Reversed phase column of octadecylsilane (ODS) C18 with isocratic mobile phase containing methanol, and phosphate buffer of pH 6 in a ratio of 70:30 (v/v). Celecoxib is used as an internal standard at a detection wavelength of 215 nm. This method separates the analytes in a total running time less than 13 min. Linearity is obtained in the calibration curve for all analytes with a R2 value of > 0.999. Furthermore, beta-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were added, either alone or combined, to prevent the crystal formation, and any unpleasant taste of etoricoxib in oral formulations. After testing both HP-β-CD and β-CD at 3% w/w for each, the results showed that HP-β-CD is more efficient in preventing the crystal formation of etoricoxib in suspensions at room temperature than β-CD is.

Development and validation of a rapid reverse-phase HPLC method for the determination of methotrexate from nanostructured liquid crystalline systems.

A reversed-phase liquid chromatography (RP-LC) method was successfully developed and validated for the determination of methotrexate in nanostructured liquid crystalline systems composed by polyether functional siloxane and silicone polyether copolymer. The LC method was performed on RP C18-ODS column, Agilent Zorbax® (4.6 x 250 mm, 5 μm), maintained at room temperature, with a mobile phase constituted by a mixture of 50 mM ammonium acetate buffer (pH 6.0) and methanol (77:23,v/v) with a flow rate of 1.0 mL/min, using ultraviolet detection at 313 nm. The parameters used in the validation process were linearity, specificity, intra and inter-day precision, accuracy, robustness. The quantitation and detection limits yielded good results. The calibration plot assumed linear behavior from 5.0-150.0 μg. mL-1 (r2 = 0.9999). The methotrexate was subjected to oxidation, acid, base and neutral degradation, photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of methotrexate. The nanostructured liquid crystalline systems did not interfere with the analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.20 %. The method developed proved to be simple, sensitive, accurate, precise, reproducible and therefore adequate for routine analysis of methotrexate in nanostructured liquid crystalline systems.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR PHENYTOIN SODIUM AND PHENOBARBITONE IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: To develop an accurate, simple, rapid, precise, and linear RP-HPLC method for the simultaneous estimation of phenytoin sodium and phenobarbitone in tablet dosage form and validated as per ICH guidelines.Methods: The method used was a reverse phase HPLC (RP-HPLC) method using Hypersil BDS C18, (250×4.6 mm, 5 μm) column, mobile phase comprising of methanol: phosphate buffer (pH 5.0) (50:50), flow rate of 1.0 ml/min and a detection wavelength of 215 nm using a UV detector.Results: The retention time for phenytoin sodium and phenobarbitone was found to be 3.97 min and 6.90 min, respectively. The linearity of developed method was achieved in the range of 10-30 μg/ml for phenytoin sodium and 3-9 μg/ml for phenobarbitone. The detection (LOD) and quantitation (LOQ) limits were 1.44 and 4.36 µg/ml for phenytoin sodium and 0.40 and 1.35 µg/ml for phenobarbitone respectively.Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of phenytoin sodium and phenobarbitone in bulk and pharmaceutical formulation. The method was validated as per ICH guidelines. Hence, the method holds good for the routine analysis of phenytoin sodium and phenobarbitone in various pharmaceutical industries as well as in academics.

Development and validation of a rapid reversed-phase HPLC method for the quantification of monoclonal antibody bevacizumab from polyester-based nanoparticles

Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to improve the monoclonal antibody-based therapy, attempts have been focused in the formulation of these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography with fluorescence detection method for the determination of bevacizumab from nanoparticulate systems, according to the International Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP-C8 column with a mobile phase composed by water-0.1% (v/v) TFA and acetonitrile-0.1% (v/v) TFA in gradient mode at a flow rate of 1mLmin−1. Results showed that the proposed method is specific, linear in the range of 10–100μgmL−1 (r2=0.9997), accurate (recovery rate 100.50±0.85%), precise at the intraday and inter-day (relative standard deviation less than 1.79%) and robust. The detection and quantification limits were calculated by specific linear calibration curve with less concentrated standard (range of 1–20μgmL−1). The LOD was 2.16μgmL−1 and LOQ was 6.55μgmL−1. This method was also successfully used, for the first time, to quantify and compare the content of bevacizumab encapsulated into poly(lactic-co-glycolic acid)-based nanoparticles before and after lyophilization.

Comparative Study of Prepared Bromelain Gel Formulations and their Evaluation by HPLC Determination

Bromelain powder (proteolytic enzyme) was formulated as a gel for topical medical application by using two preparations method; Carbople 940 was used in the first formulation and Lutrol F 127 in the second.
 The physical and pharmaceutical properties of the gel formulations including the diffusion rates through the skin in vitro were evaluated.
 The best permeability of Bromelain was obtained with the 22% Lutrol F 127 gel formulation. In addition, a simple and rapid reversed-phase HPLC method was developed to monitor the quantitative analysis during the study. The used column was C18, 5µm (25 cm length), the mobile phase consisted of 70% methanol in 0.1 M dibasic potassium phosphate. The retention time of Bromelain was 7.3 minutes and the method proved precision as the straight line relationship of peaks areas and concentrations was with a correlation coefficient 0.998, and the RSD value for five successive determinations for same sample solution was 1.2%.

A simple and rapid RP-HPLC method for the simultaneous determination of piperacillin and tazobactam in human plasma

A rapid and sensitive reverse phase HPLC (RP-HPLC) method for the simultaneous quantitation of piperacillin and tazobactam in human plasma has been developed and validated. The method utilizes a novel, simple and rapid solid phase extraction step, which results in an improved extraction yield of analytes from human plasma, as well as significantly reduced interference from serum components at low UV wavelength detection compared to previously published liquid–liquid extraction methods. Chromatographic separation was carried out on a Hypersil ODS C18, 3μm column using an acetonitrile-trifluoroacetic acid-water gradient elution with dual wavelength quantitation at 254nm for piperacillin and 218nm for tazobactam. Linear relationships between peak area and drug concentration were obtained in the range of 1.0–200μg/mL for piperacillin and 0.78–50μg/mL for tazobactam, with r2=0.9997 and 0.9994 respectively. The assay proved to be sensitive (with a lower limit of quantitation of 1μg/mL for piperacillin and 0.78μg/mL for tazobactam), specific (no interference from plasma components at either 218nm or 254nm), and reproducible (both intra- and inter- day coefficients of variation were ≤6%). With a total process/assay time of less than 30min, the method provides a simple, precise and reproducible assay for monitoring piperacillin and tazobactam plasma levels that can be readily adapted for routine clinical use.

Study of Stress Degradation Behavior of Rasagiline Mesylate Under Hydrolytic Conditions by High Performance Liquid Chromatography

A precise and rapid reverse phase HPLC method has been developed for the estimation of Rasagiline Mesylate in bulk drug under stressed hydrolytic conditions according to ICH guidelines Q1(A)R2. The stressed samples of Rasagiline Mesylate were analyzed using Prontosil ODS column. The mobile phase consists of methanol and Phosphate buffer. The pH of the phosphate buffer was adjusted to 3.0 using orthophosphoric acid. The composition of mobile phase Methanol: Phosphate buffer (75:25 v/v) was kept constant up to 1 minute and then changed to 90:10 v/v in next 9 min by HPLC gradient programming. The flow rate was maintained at 1.0 ml/min and detection was carried out at 210 nm using 996 PDA detector. The method was validated in terms of linearity, accuracy, precision specificity and selectivity. The method was found to be linear in the range of 5-15 µg/ml with a correlation coefficient of 0.993.

Determination of 5-hydroxymethyl-2-furaldehyde in royal jelly by a rapid reversed phase HPLC method

A new method for the determination of 5-hydroxymethyl-2-furaldehyde (HMF) in royal jelly by reversed phase HPLC is presented. The method is rapid and simple and it has been validated in terms of limits of detection and quantification, linearity, precision and accuracy, in addition to being tested on twenty-five real samples supplied by a local beekeeper. The method showed good limits of detection and quantification (0.53 mg kg−1 and 1.60 mg kg−1, respectively) and an excellent linearity (R2 = 0.9999). Precision values, expressed in terms of repeatability and reproducibility (3.8% and 15.6%, respectively), are acceptable according to Horwitz's theory. Average recovery values were 98 ± 2%. Preliminary data suggest that while HMF concentration in freshly harvested royal jelly and in samples stored at 4 °C is below the quantification limit, it is possible to measure quantifiable concentrations in samples stored at room temperature.

A rapid reversed-phase HPLC method for the simultaneous analysis of olanzapine and simvastatin in dual nanostructured lipid carriers

In the present work, a rapid reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of simvastatin, including the lactone prodrug (SV) and the respective active hydroxy acid form, simvastatin acid (SVA) and olanzapine (OL) in a formulation containing co-encapsulating-nanostructured lipid carriers (Combo-NLC). The desired chromatographic separation was achieved on a Phenomenex Luna Phenyl-Hexyl, 5 μm (150 × 3 mm) column at a temperature of 35 °C, under isocratic conditions using UV detection at 230 nm. The optimized mobile phase consisted of a mixture of ammonium acetate aqueous solution (0.02 M), methanol and acetonitrile (30 : 35 : 35, v/v/v) at a flow rate of 0.8 mL min−1. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.5–100 μg mL−1, with determination coefficients, R2, exceeding 0.9994 for all three compounds, SVA, SV and OL. The method was shown to be specific, without the interference of NLC components, precise at the intra-day and inter-day levels, as reflected by the relative standard deviation values, lower than 9.014%, accurate with bias not exceeding 15% and characterized by a recovery rate of 100 ± 8%. The limits of detection and quantification were, respectively, 0.07 and 0.22 μg mL−1 for OL, 0.12 and 0.36 μg mL−1 for SV and 0.09 and 0.27 μg mL−1 for SVA. The method was successfully applied for the determination of Combo-NLC entrapment efficiency.

New chiral reverse phase HPLC method for enantioselective analysis of ketorolac using chiral AGP column

A simple, specific, precise, sensitive and rapid reverse phase-HPLC method was developed for determination of ketorolac enantiomers, a potent nonnarcotic analgesic in pharmaceutical formulations. The method was developed on a chiral AGP column. Mobile phase was 0.1M sodium phosphate buffer (pH 4.5): Isopropanol (98:2, v/v), at a flow rate of 1mL/min with run time of 15min. Ultraviolet detection was made at 322nm. The linearity range was 0.02–10μg/mL for each of the enantiomers. The mobile phase composition was systematically studied to find the optimum chromatographic conditions. Validation of the method under the conditions selected showed that it was selective and precise and that the detector response was linear function of ketorolac.

Simultaneous determination of sparfloxacin and commonly used H2 receptor antagonists by RP-HPLC: application to in vitro drug interactions

Innovative, simple, economical, and rapid reverse phase HPLC method has been developed and validated for simultaneous determination of sparfloxacin (SPFX) and cimetidine, ranitidine, and famotidine (H2 receptor antagonists) in bulk material, pharmaceutical formulations, and human serum. Chromatographic system was consisting of schemed HPLC system having UV detector set at 232 nm for cimetidine and, 250 nm for famotidine and ranitidine. Purospher STAR C18 (250 × 4.6 mm, 5 μm) column was used, and the mobile phase (methanol, water and ACN 54:41:5 v/v/v pH 2.7 adjusted by phosphoric acid) was delivered at a flow rate of 1.0 ml min−1. The proposed method is specific, accurate (98.02–102.136%), precise (intra-day and inter-day variation 0.501–2.179%) and linear (R 2 > 0.999) with in the desired range 0.3125–25 μg ml−1 concentration. The detection limit and quantification limit were 0.009–0.084 and 0.030–0.255 μg ml−1, respectively. Paired t test was applied to verify the results. The anticipated method is applicable to routine analysis of SPFX and H2 receptor antagonists in pharmaceutical formulations as well as in human serum samples. The proposed method is also applied to study interaction studies of SPFX with H2 receptor antagonists.

Determination of the salivary retention of hexetidine in-vivo by high-performance liquid chromatography.

The non-antibiotic antimicrobial agent hexetidine is widely used at a concentration of 0.1% w/v as an oral rinse to reduce the number of viable microorganisms within the oral cavity. However, following use, the available concentration of hexetidine in the oral cavity declines with time, thus compromising the resultant antimicrobial activity. It is, therefore, desirable to determine the persistence of the agent in the oral cavity by quantification of the drug concentration in saliva, thus enabling prediction of its antimicrobial activity in the oral environment. A rapid reverse-phase HPLC method was therefore developed and validated for hexetidine in aqueous solution (Oraldene) and in saliva samples collected from volunteers post-rinsing with 15 mL of hexetidine oral rinse for 30s. The HPLC assay was sufficiently sensitive to accurately detect hexetidine in saliva up to 25 min after in-vivo use of a commercial oral rinse. Furthermore, it was possible to detect hexetidine below the published minimum inhibitory concentrations (MICs) for a selection of microorganisms. From these data a first-order elimination rate constant of hexetidine from the oral cavity was determined post-rinsing in each of six volunteers. The validated HPLC assay method presented is useful for the assay of hexetidine in the oral cavity both at and below MICs. The first-order elimination rate constant shows significant variation between volunteers.

Development of Reverse-Phase HPLC Method for Simultaneous Analysis of Metoprolol Succinate and Hydrochlorothiazide in a Tablet Formulation

Purpose: To develop a simple, sensitive and rapid reverse phase HPLC method for the simultaneous analysis of metoprolol succinate and hydrochlorothiazide in a solid dosage form. Methods: The drugs were analysed by a reverse phase C-18 column using 50mM di-sodium hydrogen phosphate:methanol:acetonitrile in a ratio of 525:225:250 as mobile phase. The flow rate was 1 ml/min and the compounds were detected by a UV-detector at 222 nm at a column temperature of 24 ± 2 ºC. The method was statistically validated for linearity and accuracy. Results: The retention time and drug content of metoprolol succinate and hydrochlorothiazide were 5.38 min, 96.05 % and 3.04 min., 97.64 %, respectively. Conclusion: The study shows that the developed method is simple and accurate and that it would be suitable for the simultaneous determination of metoprolol succinate and hydrochlorothiazide in pharmaceutical formulations.Keywords: Metoprolol succinate; Hydrochlorothiazide; Simultaneous analysis; High performance liquid chromatography; Tablet formulation

Determination of Individual Homologues and Total Content of Benzalkonium Chloride by Reversed-Phase High-Performance Liquid Chromatography Using a Short Butyl Column

Benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides that usually contains C-10, C-12, C-14, and C-16 homologues), commonly known as BKC, is used as a bacteriostatic agent in many household, food, and drug products. In this paper, we report a simple, rapid, robust, and stability-indicating reversed-phase HPLC method using a short butyl (C4) column for the simultaneous determination of each individual homologue content, as well as the total concentration of individual homologues in commercial bulk raw material batches of BKC samples. The chromatographic separation was performed on a 5 cm ACE C4 column with mobile phase consisting of water, acetonitrile, and potassium chloride. Even though using a short column can potentially cause some challenges to resolving certain critical pairs of peaks, we have successfully separated all of the analyte peaks (including those from stressed, degraded products) on a short column using an optimal mobile phase.

Analysis of cationic liposomes by reversed-phase HPLC with evaporative light-scattering detection

Cationic lipid-mediated drug delivery of small pharmaceutical molecules and biological molecules, such as proteins and DNA, has gained increasing popularity for many in vitro and in vivo applications. For this purpose, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is one of the most widely used and efficient cationic lipids. In this work, a simple and rapid reversed-phase HPLC method was developed for the simultaneous determination of cationic lipid DOTAP and neutral co-lipids cholesterol and phosphatidylcholine (DPPC or DSPC) as well as their degradation products in liposome-based drug formulations. Due to the poor UV absorbance of the lipids and their degradation products, an evaporative light-scattering detector (ELSD) was used to monitor the separation. The HPLC separation was achieved using a Phenomenex Luna C18 column at 50 °C by a linear gradient elution with methanol–water mobile phase at a flow rate of 2.0 mL/min. 0.1% (v/v) trifluoroacetic acid (TFA) was added into the mobile phase to enhance the retaining of the cationic lipid DOTAP. This newly developed method enabled direct analysis of liposomes without solvent lipid extraction, and was validated to be linear, precise, accurate, specific and sensitive. The limit of detection (LOD) and limit of quantitation (LOQ) were determined to be 0.15 and 0.30 μg, respectively, for all the four lipids. The method has been successfully employed in a wide range of lipid-based formulation screening, process development and stability testing. Studies of liposome samples under accelerated thermal conditions revealed that the hydrolysis of DOTAP, DPPC and DSPC followed pseudo-first-order kinetics.

Protein-loaded PLGA microparticles engineered by flow focusing: Physicochemical characterization and protein detection by reversed-phase HPLC

In the present study, a novel synthesis technique based on the flow focusing (FF) technology is investigated for the preparation of green fluorescent protein (GFP)-loaded poly( d, l-lactide- co-glycolide) (PLGA) microparticles. To our knowledge, this novel technique has never been applied to the formulation of proteins in polymeric systems. A simple, specific and rapid reversed-phase HPLC (RP-HPLC) method was validated for the determination of GFP in PLGA microparticles with the best chromatographic peak resolution, reduced run time and low cost of analysis. In order to achieve the finest GFP-loaded polymeric particles, experimental parameters mainly associated to the FF device were studied (liquid flow rate and pressure of the focusing air). Very high GFP encapsulation values (>90%) were obtained by this technique, and the electrokinetic characterization of these systems suggested that this protein was incorporated into the polymeric matrix. This study is intended to offer information on which to base the development of high molecular weight protein-loaded polymeric delivery systems prepared by FF.