Optimization Of Mobile Phase Composition Research Articles

Stability Indicating RP-HPLC Method Development and Validation for Imeglimin HCL in Pharmaceutical Dosage form

Background: Imeglimin HCL is a medication used in pharmaceutical products. To ensure its quality and effectiveness, it's crucial to have a reliable method to measure its concentration in these products. High-Performance Liquid Chromatography (HPLC) is a common technique for this purpose. Objective: This study aimed to develop and validate a precise and accurate stability indicating HPLC method for measuring Imeglimin HCL in pharmaceutical formulations. Methods: The method optimization process included selecting a suitable chromatographic column and determining the optimal mobile phase composition. A Credchrom C18 column (250mm x 4.6 mm x 5µm) was chosen, and a mobile phase consisting of Phosphate Buffer and acetonitrile (80:20, v/v) was identified as optimal. The selected conditions provided satisfactory resolution and a retention time of 2.5 minutes for Imeglimin HCL, with detection achieved at a wavelength of 241nm. Validation parameters, including linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ), were thoroughly evaluated. Precision was assessed through %RSD values for repeatability and intra-day and inter-day precision. Accuracy was determined by % recovery, with values ranging from 98.00% to 102%. Subsequently, force degradation studies were conducted to further assess the method's stability. Results: The developed RP-HPLC method exhibited excellent linearity (R^2 close to 1) and sensitivity, with LOD and LOQ values of 0.577722 μg/ml and 1.750673 μg/ml, respectively. Precision, as indicated by %RSD values, ranged from 0.994733 for repeatability to 0.988377–0.7480963 for intra-day precision and 0.988377–10.9883477 for inter-day precision. Accuracy, reflected by % recovery, was within acceptable limits (98.62–100.34%). These results, along with successful force degradation studies, confirmed the method's reliability and stability. Conclusions: In conclusion, the developed RP-HPLC method offers a sensitive, precise, and accurate means for the estimation of Imeglimin HCL in pharmaceutical formulations. Its robustness and stability make it suitable for routine analysis in pharmaceutical laboratories, ensuring the quality and efficacy of Imeglimin-containing products.

Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

Comparison of the impact of anion and cation selection onto cation exchange chromatography of model proteins

For a further improvement of ion exchange chromatography for protein purification, the optimization of mobile phase composition is an important factor. The effects of salt type selection on cation exchange chromatography (CEC) have been investigated in this work. Hereby, the significance of co-ion and displacing ion selection were compared. Therefore, the suitability of a constant cationic or anionic strength for multivalent salts for the determination of salt impact were evaluated. The retention and separation behavior of the three model proteins bovine serum albumin, lysozyme and α-chymotrypsin were investigated with varying salts in the elution buffer. For this, five different cations and five different anions were used. Hereby, the effects of anion and cation selection were analyzed independently. It was shown that a constant cationic strength is a suitable setup for the determination of salt effects on protein retention in CEC, as the resulting retention factors did not show a clear trend regarding ion valence. It was observed that for the used process conditions the anion selection could change the separation factor by up to 30% and the cation selection by up to 16%. These changes were attributed to protein specific salt effects of citrate on bovine serum albumin and magnesium on α-chymotrypsin. It was demonstrated that at constant cationic strength the co-ion selection can be of the same or even of higher importance for protein separation as the selection of the displacing ion in CEC.

Use of green-modified micellar liquid chromatography for the determination of Imidocarb dipropionate residues in food samples

Imidocarb dipropionate (IMD) is broadly utilized in veterinary medicine as an antimicrobial to cure many illnesses, particularly anaplasmosis in livestock. Consequently, the rapid determination of IMD residues in edible bovine food samples is of high significance to guarantee food safety. In the meantime, the environmental influences must be considered to reduce the hazardous impacts of control laboratories. Modifying micellar liquid chromatography (MLC) using a natural deep eutectic solvent (NADES), generated utilizing choline chloride (ChCl) and ethylene glycol (EG), was successfully applied to determine imidocarb (IMD) residues in bovine food samples including meat, liver, kidney, and milk. An experimental design approach was employed for multivariate optimization of important mobile phase factors comprising the concentration of sodium dodecyl sulfate (SDS) in mole L-1, and the percentage volumes of NADES, and ethanol (EtOH). The optimal sustainable mobile phase composition was conquered through the Box Behnken design (BBD) optimization approach considering chromatographic peak height, and peak width at 50% of its height (W 50%) as influential responses. The optimal composition of the modified micellar mobile phase was found to be 0.075 M aqueous SDS solution with Ethanol and NADES in the ratio (40:50:10) with pH adjusted to 4.0 using 0.2 M acetic acid solution. At this optimal composition, IMD was efficiently eluted within 0.8 min when the flow rate was 1 mL/min with no interference by endogenous components and proteins in bovine food samples. Pretreatment of food samples was performed by extracting with an aqueous solution of SDS surfactant only. No organic solvent was required during the extraction step. To validate the presented methodology, the regulations of the Food and Drug Administration (FDA) for bioanalytical analysis were applied. The greenness of the proposed method was estimated utilizing three measures involving the Complementary Green Analytical Procedure Index (ComplexGAPI), the Analytical GREEnness metric (AGREE), and the modern White Analytical Chemistry (WAC) assessment tool. Using these tools, a comparative study was conducted between the proposed method and previously reported liquid chromatographic methods. The proposed method was found to be the most sustainable.

Stability-Indicating Analytical Method Development Using Quality by Design (QbD) Approach for Simultaneous Estimation of Budesonide and Levosalbutamol.

Budesonide is a corticosteroid; levosalbutamol is bronchodilator. In combination, they are used in the treatment of asthma and chronic obstructive pulmonary disease. The present article portrays the development of a stability-indicating high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of budesonide and levosalbutamol using design of experiment (DOE) principles. DOE was applied in development part in which a Box-Behnken design was used to study the effect of factors on the Rf value of the drugs. Twenty-five experimental runs were performed to optimize the chromatographic conditions. Aluminum sheets precoated with silica gel 60 F254 were used as the stationary phase. The optimized mobile phase composition was found to be toluene-ethyl acetate-methanol-ammonia (4:1.8:1.8:0.2) and saturation time 20 min, quantified by densitometric analysis at 231 nm. Moreover, the drugs were subjected to acid and alkali hydrolysis, oxidation, thermal, and photodegradation. The drugs undergo degradation under mainly acidic and basic conditions. Also, the degraded products were well resolved from the pure drug with significantly different Rf values. Linearity was performed in the ranges of 224-1120 ng/band for budesonide and 280-1400 ng/band for levosalbutamol. The method was validated according to the International Conference on Harmonization guidelines. This approach is useful to expedite method development and optimization activities in analytical laboratories. The experimental data revealed that the volume of toluene and methanol in the mobile phase significantly affected the Rf value of both the drugs, and ammonia significantly affected the Rf value of levosalbutamol.

Fractionation of secondary metabolites from Serratia plymuthica UBCF_13 based on polarity properties

Serratia plymuthica UBCF_13 bacteria produce compounds capable of suppressing the growth of various pathogenic fungi. There are several types of compounds produced by these bacteria. The compounds produced by S. plymuthica bacteria have different abilities and characteristics. So, to determine the capabilities and characteristics of each compound, a compound separation technique or method is needed. This study was conducted to determine the optimal mobile phase composition for the separation of compounds. Optimization of the mobile phase was carried out by spotting the extracellular extract of S. plymuthica on thin layer chromatography (TLC) using several comparisons of ethyl acetate with hexane and ethyl acetate with methanol. The separation technique in this research will be useful for further study of the antifungal compound Serratia plymuthica UBCF_13, such as the identification and characterization of each of the compounds it produces. In this study, the best mobile phase was not found in the separation of compounds. There are several mobile phases that separate the compounds, for further test the elution gradient method is used.

Development of a novel method for polar metabolite profiling by supercritical fluid chromatography/tandem mass spectrometry

Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from –5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples.

Validation of High-Performance Liquid Chromatography for Determination of Bromelain in Pineapple (Ananas comosus (L) Merr) Water

Objective: The aim of the present study was to validate HPLC method for analysis bromelain levels in pineapple water and application that method to determine the storage time of pineapple water. Methods: The reversed phase of HPLC method was tested and optimized before it is validated. The composition and the flow rate of the mobile phase is the optimized parameter. The analytical parameters validated were detection limits, linearity, accuracy and precision. Pineapple water was stored for 8 h at 10ºC and bromelain was determined using the validated HPLC method. Result: The optimum mobile phase composition was methanolwater (70:30) with a flow rate of 1 ml/min. The detector concentration-response was linear with coefficient of determination of 0.993. The accuracy of HPLC method at a recovery of 1 and 2% bromelain was 106.37 ± 1.94% and 98.12 ± 1.29% (n = 5), respectively. The precision, expressed as the coefficients of variation (CV), at 1 and 2% bromelain were 1.83 and 1.32% (n = 5), respectively. Bromelain level at zero time was 81.53%. After storage for 8 h at 10ºC, bromelain levels in pineapple juice appeared to decrease not statistically significant (p > 0.05), with to mean value of 78.46 ± 2.88%. Conclusion: The HPLC method developed was valid to analyze accurately concentrations of bromelain in pineapple water and it can be used to study the shelf life of pineapple water based on bromelain content. The bromelain content in pineapple juice was not statistically significantly different after 8 h storage at 10°C.

Off-line comprehensive size exclusion chromatography × reversed-phase liquid chromatography coupled to high resolution mass spectrometry for the analysis of lignocellulosic biomass products

Biochemical and thermochemical processes are two pathways to convert lignocellulosic biomass into fuels and chemicals. Both conversion types produce aqueous complex samples containing many oxygenated chemical functions over a wide range of masses. Nowadays, composition of these biomass products is still largely unknown, especially their nonvolatile part (300–1000 Da) mostly made of carbohydrates and their derivatives. In the present study, size exclusion chromatography (SEC) was investigated and applied on water soluble phase of a fast pyrolysis bio-oil (thermochemical conversion) and on aqueous phase of pretreated wheat straw (biochemical conversion). An optimization of mobile phase composition using model molecules was necessary to limit non-steric interactions and elute all chemical families. At the end, separation of carbohydrates, heterosides and aromatic species was performed. The chemical organization of SEC chromatograms was confirmed by coupling SEC with a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) using electrospray ionization (ESI) in the negative mode. On-line SEC-UV/FT-ICR MS hyphenation was a powerful tool to provide exact mass distribution of samples and get molecular formulae classed by chemical family. To go further, the complementarity of SEC with reversed-phase liquid chromatography (RPLC) was established with an off-line comprehensive 2D-LC analysis of the two samples. First, 140 fractions were collected physically from SEC separation for each sample, then each fraction was analyzed by RPLC hyphenated to an Ion Trap - Time of Flight mass spectrometer (SEC × RPLC-UV/IT-TOF MS) using ESI in both positive and negative modes. This comprehensive approach combining 2D-LC and high resolution mass spectrometry nearly doubled the number of peaks detected in comparison with 1D RPLC analysis and thus offered well resolved 2D contour plots, considered as relevant analytical fingerprints of the aqueous phase of biomass samples.

A simple, rapid and fully validated HPLC method for simultaneous quantitative bio-analysis of rosuvastatin and candesartan in rat plasma: Application to pharmacokinetic interaction study.

A simple, precise and accurate HPLC method was developed, optimized and validated for simultaneous determination of rosuvastatin and candesartan in rat plasma using atorvastatin as an internal standard. Solid-phase extraction was used for sample cleanup and its subsequent optimization was carried out to achieve higher extraction efficiency and to eliminate matrix effect. A quality by design approach was used, wherein three-level factorial design was applied for optimization of mobile phase composition and for assessing the effect of pH of the mobile phase using Design Expert Software. Adequate separation for both analytes was achieved with a Waters C18 column (250 × 4.6 mm, 5 μm) using acetonitrile-5 mm sodium acetate buffer (70:30, v/v; pH adjusted to 3.5 with acetic acid) as a mobile phase at a flow rate of 1.0 mL/min and wavelength of 254 nm. The calibration curves were linear over the concentration ranges 5-150 and 10-300 ng/mL for rosuvastatin (ROS) and candesartan (CAN), respectively. The validated method was successfully applied to a pharmacokinetic study in Wistar rats and the data did not reveal any evidence for a potential drug-drug interaction between ROS and CAN. This information provides evidence for clinical rational use of ROS and CAN.

Controlling selectivity of polymer-based monolithic stationary phases.

In this work, we aimed to prepare a monolithic capillary column that allowed an isocratic separation of ten dopamine precursors and metabolites in a single run. Segments of five zwitterion sulfobetaine polymer monoliths have been modified by zwitterion phoshorylcholine by using an ultraviolet-initiated two-step photografting. Columns with 0, 33, 50, 66, and 100% of modified length were prepared. Effect of length of the modified segment and mobile phase composition has been tested. All columns provided dual-retention mechanism with reversed-phase retention in highly aqueous mobile phase and hydrophilic interaction mechanism in highly organic mobile phase. The retention mechanism was controlled by the composition of the mobile phase and has been described by a three-parameter model. We have used regression parameters to characterize the retention of analyzed compounds and to study individual pathways of dopamine metabolism. Comprehensive optimization of mobile phase composition allowed to find an optimal composition of the mobile phase and stationary phase surface chemistry arrangement to achieve desired separation. Optimized columns provided an isocratic separation of all tested compounds in less than nine min.

Effect of the Mobile Phase Composition on Selectivity in Supercritical Fluid Chromatography in the Separation of Salbutamol Enantiomers

Three chiral chromatography stationary phases based on amylose and cellulose were tested for the supercritical fluid chromatography separation of antiasthmatic drug salbutamol enantiomers. A cellulose tris- 3-chloro-4-methylphenylcarbamate stationary phase provided the highest selectivity for this separation. The optimization of mobile phase composition allowed us to maximize the selectivity coefficient and to minimize the peak asymmetry factors. A successful salbutamol enantiomer separation is achieved at moderate modifier fraction (10–15 vol %) in the mobile phase on this column. Amine additives are necessary to suppress peak tailing. Among three tested amines isopropylamine gives the most symmetrical peaks along with the highest enantioselectivity.

Degradation of ouabain in 80-year-old injection solution studied by HILIC–MS

An 80-year-old injection solution of ouabain was analyzed using HPLC–MS. A new separation method involving hydrophilic interaction liquid chromatography (HILIC) using an amide-based stationary phase with mass spectrometric detection was developed for the analysis. The optimized mobile phase composition was acetonitrile:water 85:15 (v/v) at the flow rate of 0.2 cm3/min under the isocratic elution. The ouabain content was found reduced to 56% of declared content and probable degradation product with oxidized hydroxy group was established. The ESI-MS2 fragmentation mechanism of ouabain was proposed.

Simultaneous optimization of mobile phase composition and pH using retention modeling and experimental design

Chromatographic methods are progressing continuously. Increasing sample complexity and safety expectations lead to higher regulatory demands, hence challenges in liquid chromatography analysis are rising, even today, when faster and faster chromatographic systems are extensively employed and become widely accessible for successful method development.The goal of this study was to investigate the impact of mobile phase influences as important factors of selectivity tuning in method development. This would mitigate mobile phase-related robustness issues throughout the method’s lifecycle.To discover and understand these effects, a new module of chromatographic modeling software DryLab (ver. 4.3.4. beta) was introduced and a special experimental design (DoE) was tested, allowing the simultaneous optimization of solvent-dependent parameters, such as gradient time (tG), ternary eluent composition (tC) and pH, requiring 18 input experiments (2 × 3 × 3 = 18).Additionally, the model creation, using a UPLC system and a narrow bore column (50 × 2.1 mm), the entire experimental work could be finished in 2–3 hours. To demonstrate the applicability of this new design, amlodipine and its related pharmacopoeia impurities (A–H) were subjected to be used in a case study. Predicted vs. Experimental (or Verification) runs showed excellent agreement, average retention time deviations were typically less than 1 s. Modelled robustness testing was also performed, elucidating all important mobile phase and instrument parameters that could influence a method’s lifetime performance. Furthermore, as the in silico robustness testing is the least time consuming part of the method development process, it can be used extensively to evaluate robustness even at the very early part in stage 1 of the Method Life Cycle (MLC).

Ultrafast monolithic HPLC method for simultaneous quantification of the anticancer agents, imatinib and sorafenib: Application to tablet dosage forms

Purpose : To develop and validate a simple ultrafast monolithic high performance liquid chromatography (HPLC) method for the simultaneous quantification of two anti-cancer agents, imatinib and sorafenib, in pure form and tablet preparations. Method : Chromatographic separation was accomplished using Chromolith flash RP-18 HPLC-column (25 - 4.6 mm; macropores, 2 μm; mesopores, 13 – 15 nm). The optimum mobile phase composition of ammonium acetate buffer (10 mM, pH 8.5) and methanol at ratio of 35:65 v/v was used. Effluent flow rate was adjusted to 1.0 mL/min and the analysis was performed at 250 nm wavelength. The developed method was evaluated for specificity, linearity, precision and accuracy. Results : The method offered a linear relationship over the concentration range of 1 - 16 μg/ml (correction coefficient, R 2 = 0.9999) for both analytes. Limit of detection (LOD) was 0.1891 and 0.1888 μg/ml while limit of quantification (LOQ) was 0.6303 and 0.6294 μg/ml for imatinib and sorafenib, respectively. Mean recovery was within 100 ± 2 %. The utility of the new method was demonstrated by its successful use for the analysis of commercially available tablet formulations of both drugs. Conclusion : The developed method is fast and economical, and is being recommended for routine analysis of imatinib and sorafenib in bulk drug and tablet dosage forms in quality control laboratories. Keywords : RP-HPLC, Chromolith, Imatinib, Sorafenib, Validation, Quality control

Simultaneous analysis of 310 pesticide multiresidues using UHPLC-MS/MS in brown rice, orange, and spinach

In this study, we developed a multiresidue method for the analysis of 310 pesticides in representative agricultural produce (brown rice, orange, and spinach) using ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) combined with a modified QuEChERS procedure. The optimal mobile phase composition (Methanol containing 5 mM ammonium formate and 0.1% formic acid) produced high sensitivity and reliable results. Also, the relationship between injection volume and repeatability of peak area was investigated. Most of the target pesticides had a limit of quantitation under 10 ng g−1, and correlation coefficients (r2) > 0.99 in matrix-matched standards within the range of 1–100 ng g−1. To validate the optimized method, recovery tests were performed with each of the crops at 10 and 50 ng g−1 spiking levels (n = 5). Satisfactory recoveries were achieved showing that 86.8–88.7% (at 10 ng g−1) and 91.9–96.1% (at 50 ng g−1) of the pesticides met the validation criteria (recoveries in the range of 70–120% and relative standard deviation ≤ 20%). Fifteen compounds were found to show a loss of recovery due to adsorption by primary and secondary amine or graphite carbon black. In the case of brown rice, 86.1% of pesticides showed an insignificant matrix effect (<±20%), while 35.2% and 41.6% of pesticides in orange and spinach were in that range, respectively. Sixteen apple samples from local markets were analyzed to evaluate the applicability of the optimized method. Nineteen pesticides were detected, of which the concentrations were lower than the maximum residue limit.

Development and Validation of a Stability-Indicating HPLC Method for Imidapril and Its Degradation Products Using a Design of Experiment (DoE) Approach.

The present work focused on the application of design of experiment (DoE) principles to the development and optimization of a stability-indicating method (SIM) for the drug imidapril hydrochloride and its degradation products (DPs). The resolution of peaks for the DPs and their drug in a SIM can be influenced by many factors. The factors studied here were pH, gradient time, organic modifier, flow rate, molar concentration of the buffer, and wavelength, with the aid of a Plackett-Burman design. Results from the Plackett-Burman study conspicuously showed influence of two factors, pH and gradient time, on the analyzed response, particularly, the resolution of the closely eluting DPs (DP-5 and DP-6) and the retention time of the last peak. Optimization of the multiresponse processes was achieved through Derringer's desirability function with the assistance of a full factorial design. Separation was achieved using a C18 Phenomenex Luna column (250 × 4.6 mm id, 5 µm particle size) at a flow rate of 0.8 mL/min at 210 nm. The optimized mobile phase composition was ammonium-acetate buffer (pH 5) in pump A and acetonitrile-methanol (in equal ratio) in pump B with a run time of 40 min using a gradient method.

Controlling cell adhesion in antibody purification by expanded bed adsorption chromatography

Abstract This study introduces an improved approach for determining the most suitable mobile phase maximizing repulsion between biomass and adsorbent to reduce biomass fouling in expanded bed adsorption (EBA) chromatography. Extended DLVO theory (xDLVO) has been utilized to comprehensively calculate optimal mobile phase compositions, thus increasing efficiency, improving hydrodynamics as well as product recovery. Biomass-adsorbent interaction energy surfaces were generated as a function of pH, ionic strength, and salt type, investigating the interactions of CHO cells with MabDirect® Protein-A beads. We have shown that mobile phases consisting of Na2SO4 were best suited at low ionic strength, whereas NaCl was more favorable at higher ionic strength. This was found to increase product recovery while maintaining a constant level of purity of monoclonal antibodies. Our method provides a tool for rapid method development of EBA purifications that can be dynamically adapted to operational requirements. In our case, the resulting optimized process was able to outperform the current protocol by up to 10% in terms of product recovery.

Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.

Separation of some phenolic acids in micellar liquid chromatography using design of experiment-response surface methodology

Derringer’s desirability function combined with face-centered cube central composite design was used to map the chromatographic response surface for simultaneous optimization of resolution and analysis time of four phenolic acids (vanilic, cafeic, para-coumaric and ferulic) in micellar liquid chromatography. The factors studied were the concentration of sodium dodecyl sulfate (SDS), volume percentage of propanol (PrOH), and acetic acid (AcOH) in mobile phase. The chromatographic response functions (CRFs) were formed using two sigmoidal desirability functions. The quality of the obtained chromatograms in each experiment was calculated using CRF, and the model was built by the use of multiple linear regression (MLR) to correlate the obtained CRFs with the experimental factors and their interactions. The developed MLR model showed good descriptive and predictive ability (R2 = 0.9569 and F = 24.45). The optimum mobile phase composition was found to be [SDS] = 20 mM, 1.0% (v/v) PrOH and 2.0% (v/v) AcOH, which provided good resolution and analysis time for the separation of target phenolic acids. The efficiency of prediction of polynomial model was confirmed by performing the experiment under the predicted optimal conditions.