C18 HPLC Research Articles

Stability Indicating HPLC Method Development and Validation for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Pharmaceutical Dosage Form

In the present work, simple, accurate and rapid stability indicating RP-HPLC method has been developed for the simultaneous estimation of Metformin and Empagliflozin in bulk and tablet dosage form. Separation of analytes was carried out on Ascentis C18 HPLC column (4.6 x 150 mm, 5 μm) using a mobile phase of 0.1% orthophosphoric acid and acetonitrile (60:40, v/v), at a flow rate of 1.0mL/min and measurement carried out at UV 260 nm. The validation parameters namely linearity, sensitivity, selectivity, specificity. robustness, precision and accuracy were determined. The peak area response-concentration curve was linear, over the concentration range of 125-750 μg/mL for Metformin and 1.25-7.50 μg/mL for Empagliflozin with quantitation limits of 4.59 μg/mL for Metformin, and 0.08 μg/mL for Empagliflozin. The proposed method was successfully validated for the determination of Metformin and Empagliflozin simultaneously in combined tablet dosage form. The performance of the present method was compared with available RP-HPLC methods and found to be rapid and economical. The proposed stability-indicating RP-HPLC method can be successfully applied to quality control and drug analysis.

A Stability Indicating Method Development of Lopinavir and Rotinavir in Combined Tablet Dosage Forms by RP-HPLC

A new stability indicating RP-HPLC method was developed for simultaneous estimation of Lopinavir and Rotinavir in combined tablet formulations. Chromatography was carried out on a Kromasil C18 HPLC Column (250 x 4.6mm; 5µm) eluting with a mobile phase consisting of a 30:70v/v mixture of Phosphate buffer and acetonitrile at a flow rate of 1.0mL/minute. The wavelength detection was fixed at 220nm. Using standard addition method accuracy was assessed. The developed HPLC method was validated with respect to precision, specificity, accuracy, linearity and robustness. Forced degradation studies on the formulation were conducted by adopting the proposed method to assess the stability of the analytes under acid, base, peroxide, thermal and photolytic conditions and suitability of the method to resolve the degradation products.

Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release.

This paper discusses the development and validation of a rapid method for the reversed phase HPLC-UV quantification of biodegradable poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres co-loaded with two neuroprotective agents (dexamethasone and melatonin) (DX-MEL-MSs) to be intravitreally administered as a promising glaucoma treatment. The study was performed to validate two procedures that quantify the content of the two active substances entrapped into the polymer matrix during an encapsulation efficiency assay and the amount of drugs liberated over time during the in vitro release assay. The reversed-phase method allowed for the simultaneous determination of dexamethasone and melatonin, which were respectively detected at 240.5 and 222.7 nm. Chromatographic separation was performed using an Ascentis® C18 HPLC Column (25 cm × 4.6 mm, 5 µm) with an isocratic mobile phase composed of methanol-water (70:30, v/v) with 1.0 mL min−1 flow rate. The two procedures were validated analytically in terms of system suitability testing, specificity, linearity, precision, accuracy, sensitivity, and robustness. Both the validated procedures were applied to characterize DX-MEL-MSs and were found appropriate to quantify the drug quantities encapsulated and estimate their release profile over 10 days. The validation study designed in this work can be helpful for planning any other protocols that refer to the quantification of PLGA based drug delivery systems.

Experimental Design Approach for Quantitative Expressions of Simultaneous Quantification of Two Binary Formulations Containing Remogliflozin and Gliptins by RP-HPLC

The aim of this study was to develop a fast RP-HPLC method for simultaneous measurement of two antidiabetic formulations (vildagliptin + remogliflozin and teneligliptin + remogliflozin) under identical experimental conditions. Using the Box–Behnken approach and response surface design, the interaction and quadratic influence of three variable parameters, acetonitrile %, pH of the mobile phase, and flow rate, on resolution between the peaks were optimized. To forecast the resolution of peaks (2.7 and 6.5) for the three anti-diabetic medications, the design space with desirability function was used to find the optimal chromatographic conditions. Isocratic elution with 58:42 acetonitrile and phosphate buffer (20 mM KH2PO4, pH adjusted to 4.9 with orthophosphoric acid) over a Zorabx C18 HPLC column with a flow rate of 1.2 mL min−1 separated all three analytes in 2.5 min. In addition, the optimized HPLC process was validated using ICH recommendations. The devised HPLC method’s precision and accuracy were proven by the low percent relative standard deviation (0.60–1.65%), good percentage recovery (98.18–101.50%), and low percentage relative errors (0.20–1.82%). The method’s robustness was also proven by slightly varying the five separate parameters. Finally, the accuracy of the proposed HPLC approach was confirmed using a standard addition method for simultaneous determination of vildagliptin + remogliflozin and teneligliptin + remogliflozin from formulations. Furthermore, the findings demonstrated that experimental design can be successfully used to optimize chromatographic conditions with fewer runs. The devised HPLC method for simultaneous quantification of two binary combinations utilizing the same chromatographic conditions is fast, accurate, precise, and easy, and it might be utilized in laboratories for routine quality control investigations on both formulations.

Features of assay and decomposition dynamics of 2-methoxy-4-(1-propenyl)hydroxybenzene in biological material

The objective was to study the features of assay and dynamics of decomposition of 2-methoxy-4-(1-propenyl)hydroxybenzene in biological material. Extraction, semi-preparation chromatography, TLC, HPLC, GC-MS and UV-spectrophotometry were used as test methods. 2-Methoxy-4-(1-propenyl)hydroxybenzene was extracted from the biological material by double infusion (45 min each) with ethyl acetate at a 2:1 mass ratio of isolating agent and biomatrix. Purification was performed by extraction and chromatography in a semi-preparative (190×10 mm) L 40/100 μm silica gel column using a hexane-dioxane (7:3) eluent. The analyte was determined by TLC methods (Sorbfil plates, hexane-acetone 9:1 as a mobile phase), HPLC [Discovery C18 HPLC Column (250×4.6 mm), acetonitrile-acetate buffer pH 5.5 (5:5) as a mobile phase], GC-MS [DB-5MS EVIDEX (25 m×0.2 mm) column with 5%-phenyl-95% dimethyl polysiloxane as a stationary phase], UV-spectrophotometry (95% ethanol as a solvent). The proposed assay method for 2-methoxy-4-(1-propenyl)hydroxybenzene in biomaterial (liver tissue) is validated for linearity, selectivity, accuracy and precision. The study results showed that the decomposition rate of the analyte increases as the store temperature increases. At 0-2 °C, 8-10 °C and 18-22 °C 2-methoxy-4-(1-propenyl)hydroxybenzene is stable for 480, 390 and 260 days respectively.

Zsírban oldódó A-, D2-, D3-, E- és K3-vitaminok meghatározása izotóphígítással és LC-MS/MS műszeregyüttessel

The purpose of our publication is the determination of the total amount (of natural origin and added) of fat-soluble vitamins A, D2, D3 and E in low amounts in foods (wheat flour, soft drinks, effervescent tablets) and dietary supplements using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The samples were diluted with isotope-labeled derivatives of the target components (vitamin A-d6, vitamin D2-d3, vitamin D3-d3, vitamin E-d6), and after extraction and saponification, they were purified by liquid-liquid extraction. After a solvent exchange, the concentration of the vitamins was determined on a C8 HPLC column using acidic mobile phases (0.1% formic acid in water/methanol) and LC-MS/MS technique. In dietary supplements, the analysis of the fat-soluble vitamin K3 may also be important, because the use of vitamin K3 is currently not approved in human formulations. During the determination of vitamin K3, saponification is not necessary, due to its structure, alkaline hydrolysis would lead to the decomposition of vitamin K3, so this component was analyzed by a method different from the one used for the other vitamins. LC-MS/MS analysis of small amounts of vitamin K3 is more complicated than that of other vitamins due to the low sensitivity of the MS instrument to vitamin K3. The determination of vitamin K3 was therefore carried out after chemical derivatization with L-cysteine as a derivatizing reagent, also with isotope dilution and LC-MS/MS technique. After intralaboratory validation, the methods were successfully used in domestic and international proficiency tests in infant formulas and liquid vitamin preparations.

A saponin-polybromophenol antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA polymerase

BackgroundGradual increase of multidrug resistant infections is a threat to the human race as MDR plasmids have acquired.>10 mdr and drug efflux genes to inactivate antibiotics. Plants secret anti-metabolites to retard growth of soil and water bacteria and are ideal source of antibiotics. PurposePurpose of the study is to discover an alternate phyto-drug from medicinal plants of India that selectively kills MDR bacteria. MethodsMDR bacteria isolated from Ganga river water, milk, chicken meat and human hair for testing phyto-extracts. Eighty medicinal plants were searched and six phyto-extracts were selected having good antibacterial activities as demonstrated by agar-hole assays giving 15 ​mm or greater lysis zone. Phyto-extracts were made in ethanol or methanol (1:5 w/v) for overnight and were concentrated. Preparative TLC and HPLC were performed to purify phytochemical. MASS, NMR, FTIR methods were used for chemical analysis of CU1. In vitro RNA polymerase and DNA polymerase assays were performed for target identification. ResultsCU1 belongs to a saponin bromo-polyphenol compound with a large structure that purified on HPLC C18 column at 3min. CU1 is bacteriocidal but three times less active than rifampicin in Agar-hole assay. While in LB medium it shows greater than fifteen times poor inhibitor due to solubility problem. CU1 inhibited transcription from Escherichia coli as well as Mycobacterium tuberculosis RNA Polymerases. Gel shift assays demonstrated that CU1 interferes at the open promoter complex formation step. On the other hand CU1 did not inhibit DNA polymerase. ConclusionPhyto-chemicals from Cassia fistula bark are abundant, less toxic, target specific and may be a safer low cost drug against MDR bacterial diseases.

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF AZELNIDIPINE AND TELMISARTAN IN TABLET DOSAGE FORM BY RP- HPLC

A simple, accurate and precise method for the simultaneous determination of azelnidipine and telmisartan in bulk drug and pharmaceutical dosage has been developed by RPHPLC method. Separation was performed on a Hyperchrom ODS C18 HPLC Column (250*4.6mm) column and Buffer 0.05M Potassium dihydrogen orthophosphate (KH₂PO₄) Buffer (pH-4.0): Methanol (60:40) as a mobile phase, at a flow rate 1ml/min and UV detection wavelength 215 nm. The calibration of the method was performed by concentration range of 20-60μg/ml for telmisartan and 40-120 μg/ml for azelndipine. The validation of proposed method was carried out for accuracy, precision, ruggedness, specificity for both alzenidipine and telmisartan the method can be used for routine quality analysis of titled drug in tablet formulation.

New HPLC-UV analytical method for quantification of metronidazole: Application to ex vivo ocular kinetic assessments following the administration of thermosensitive ocular in situ gel

Metronidazole eye drops have been used to treat Acanthamoeba keratitis. However, ophthalmic preparations also have some limitations, one of which is the rapid elimination of the drug, that reducing the effectiveness of the drug. Accordingly, an alternative delivery approach can be applied to overcome this issue. Additionally, as one of critical steps in the formulation development, analyitical methods that allow the quantification of metronidazole in ex vivo corneal permeation and deposition should also be developed. Here, we report a validated high-performance liquid chromatography method (HPLC-UV) according to ICH guidelines for the measurements of metronidazole concentrations following formulation of thermosensitive ocular in situ gel and its administration in ex vivo porcine corneas. The development of extraction techniques and optimization of HPLC conditions were optimized using analytical quality by design. Xselect™ CSHTM C18 HPLC column (Water, 3.0 × 150 mm, particle size 3.5 m) was used to separate all analytes by isocratic elution with mobile phases of acetate buffer and acetonitrile with LLOQ value of 0.08 μg/mL. The resulting method proved to be selective, precise, and accurate and was succefully applied to determine ocular kinetic profiles of metronidazole from thermosensitive ocular in situ gel in ex vivo porcine corneas, showing that this approach was able to improve the concentration of metronidazole in the corneal tissues. We, therefore, suggested that HPLC-UV approach developed in this study has the potential to be used in drug release evaluation, therapeutic drug control research, ocular kinetics, and toxicological evaluation.

Simultaneous estimation of pioglitazone, glimepiride & metformin hydrochloride in bulk & tablet dosage form by UV, RP-HPLC method

A stability-indicating UV spectroscopic and high-performance liquid chromatography (RP-HPLC) method is developed for the quantification ofPioglitazone, Glimepiride & Metformin Hydrochloride drug substances. UV spectroscopic method was developed and validated, the wavelength selected for simultaneous estimation were 226nm for pioglitazone, 229nm for glimepiride and 232nm for metformin hydrochloride. The isosbestic point found for the analysis was 229nm. Selected mobile phase was a combination of methanol and water with a ratio of 70% Methanol and 30 % HPLC water with the flow rate of 0.85ml/min. The analyte was analysed on the C18 HPLC column having the pore size of 5 microns at room temperature. The method is validated according to ICH guidelines, the retention time of about 4.0min for metformin, 5.5min for Pioglitazone and 6.8min for Glimepiride was observed. The linearity range with regression co-efficient for Pioglitazone, Glimepiride & Metformin Hydrochloride is 3-15 μg/mL,0.4-1.2 μg/mL and 100-500 μg/mL and 0.9998, 0.9991, 0.9991 respectively.

Pharmacokinetics of genistein distribution in blood and retinas of diabetic and non-diabetic rats

Genistein, a natural tyrosine kinase inhibitor, may act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by its nonlinear pharmacokinetics. We aimed to determine genistein's kinetics and retinal tissue distributions in normal and diabetic rats. We developed an isocratic, reverse-phase C18 HPLC system to measure genistein concentration in blood and retinas of streptozotocin (65 mg/kg IV)-diabetic and non-diabetic rats receiving two types of genistein-rich diet (150 and 300 mg/kg) for ten days. Genistein's decay exhibited a two-compartmental open model. Half-lives of distribution and elimination were 2.09 and 71.79 min, with no difference between groups. Genistein steady-state concentration in blood for 150 and 300 mg/kg diet did not differ between diabetic (0.259 ± 0.07 and 0.26 ± 0.06 μg/ml) and non-diabetic rats (0.192 ± 0.05 and 0.183 ± 0.09 μg/ml). In retina, genistein concentration was significantly higher in diabetic rats (1.05 ± 0.47 and 0.997 ± 0.47 μg/gm wt. vs. 0.087 ± 0.11 and 0.314 ± 0.18 μg/gm wt., p < 0.05). The study determined that increasing genistein dose did not change its bioavailability, perhaps due to the poor aqueous solubility. The retina's increased genistein could be due to increased permeability of blood-retinal barrier that occurs early in diabetes.

Efficacy of Different C18 HPLC Analytical Columns in the Analysis of Fumonisins B1 and B2 in Different Matrices

Fumonisins B1 and B2 are carcinogenic and commonly contaminate corn and corn-based products. Analysis of such toxins using C18 HPLC column is officially accredited but still unknown if all column types can effectively separate FB1 and FB2 or not. The present study evaluated the efficiency of 5 analytical columns with different dimensions, particle sizes, and porosities to determine these toxins in both agar cultures of Fusarium verticillioides and cornflakes. Interestingly, the traditional column 150mm of length with 5µm porous particles had close retention times to those of the short-fused core column 75mm of length with 2.7 µm reflecting in time and solvents saving. Using Sep-Pack C18 for clean-up played an important role in enhancement the limit of quantification (LOQ) for cornflake samples (5-13.7 and 16.1-39 µg kg-1 for FB1 and FB2, respectively). However, it was relatively higher for fungal culture samples that were not passed through the cleaning-up step (11.5-16 and 28.1-46.3 µg kg-1 for FB1 and FB2, respectively). Overall, the lowest LOQ was obtained using the shorter fused core column. Finally, using such clean-up in the extraction of FB1 and FB2 from spiked cornflakes samples gave good recoveries (&gt;80%) using all tested columns.

New and sensitive HPLC-UV method for concomitant quantification of a combination of antifilariasis drugs in rat plasma and organs after simultaneous oral administration.

A combination treatment comprising ivermectin (IVM), albendazole (ABZ) and doxycycline (DOX) is often prescribed for lymphatic filariasis patients. Nevertheless, there has not been an analytical method established and documented to determine these compounds simultaneously. Herein, we report a new high-performance liquid chromatographic method coupled with a UV detector (HPLC-UV) to quantify these drugs in plasma and organs. This developed analytical method was validated according to the International Conference on Harmonization (ICH) and US Food and Drug Administration (FDA) guidelines. The validated method was successfully employed to analyze IVM, ABZ along with its metabolites (albendazole sulfoxide (ABZ-OX) and albendazole sulfone (ABZ-ON)), and DOX in the plasma and organs of Wistar rats after simultaneous oral administration. An Xselect CSH™ C18 HPLC column was utilized as a stationary phase, with a mobile phase consisting of 0.1% v/v trifluoracetic acid in water and acetonitrile with a run time of 20 min. The calibration curves in biological samples were found to be linear across the concentration range of 0.01-5 μg mL-1 for IVM, ABZ and ABZ metabolites, and 0.025-10 μg mL-1 for DOX with an R value ≥0.998 in each case. The validated method was found to be selective, precise and accurate. Finally, the method developed in this study was deployed to assess the pharmacokinetic profiles and biodistribution of the combination of drugs after oral administration to Wistar rats. The validated HPLC-UV method in this study provides an extensive range of prospective applications for pharmacokinetic-based studies, therapeutic drug monitoring and toxicology.

Effect of piperine and its analogs on pharmacokinetic properties of sorafenib tosylate: Bioanalytical method development and validation

Sorafenib tosylate (ST) is a drug of choice for the treatment of advanced hepatocellular carcinoma. The bioavailability of ST varies from 29% to 38% due to the presence of food, medication, and other factors. Moreover, the dose of ST (400 mg twice a day) is also higher due to its poor bioavailability. Piperine is known as a natural bioenhancer, and the use of a bioenhancer ultimately leads to a synergistic effect. In this study, the development and validation of a precise, accurate, and reproducible bioanalytical method was carried out using reversed-phase high-performance liquid chromatography (RP-HPLC). The objective of the study is to determine the effect of piperine and its analogs on the pharmacokinetics of ST using rats. The dose of ST alone and in combination with piperine and its analogs, given orally to male Wistar rats in suspension form, and the drug plasma concentration was examined using a validated RP-HPLC bioanalytical method. The separation of piperine and ST was achieved on the Chromatopak basic C18 HPLC column with the dimension of 250 mm × 4.6 mm × 5 μm, guarded along with the phenomenex guard column using ACN:Water:Formic Acid (70:29.5:0.5 v/v) as the mobile phase at 280 nm (isosbestic point of piperine and ST). The pharmacokinetic data of ST, along with piperine, exhibit a significant increase in area under the curve (AUC) of ST (1.66-fold), while the two derivatives of piperine (5a and 5d) exhibited almost double the increase in AUC compared to piperine (2.58 and 2.42-fold, respectively).

A Validated Stability-indicating RP-HPLC Method for Piperine Estimation in Black Pepper, Marketed Formulation and Nanoparticles

Abstract: Aim: The aim of the study was to develop and validate a simple stability indicating reversephase High Performance Liquid Chromatography (RP-HPLC) method for quantitative analysis of piperine in Ayurvedic marketed formulation, black pepper and cubosome nanoformulation. Methods: The method was established by using Luna C18 HPLC column using a mobile phase consisting of acetonitrile: 0.01% ortho phosphoric acid (60:40, v/v; pH 3), delivered isocratically with flow rate of 1 mL/min and detected at 340 nm. The validation of chromatographic parameters and stress testing were performed in accordance with International Conference on Harmonization (ICH) guidelines. Results: The developed method was observed to be specific, linear (r2 > 0.999) over the selected range of concentration 0.5 to 20 μg/mL, precise (percentage relative standard deviation < 2%), with the detection and quantification limit as 0.015 and 0.044 μg/mL respectively. The relevancy of the developed method was analyzed on the piperine entrapped cubosome nanoformulation, which was formulated by fragmentation technique. The entrapment efficiency of piperine for prepared cubosome was observed to be 87.01 %. The method was implemented for the estimation of piperine in black pepper. The concentration of piperine in marketed formulation was found to be similar with the labeled concentration. The analyte peak was found to be complete resolved without any interference of additives and degrading products. Conclusion: The validated method was observed to be specific, sensitive and sufficient for the routine analysis of food products, marketed formulations and nanoparticles containing piperine.

Regioisomeric and enantiomeric analysis of primary triglycerides in human milk by silver ion and chiral HPLC atmospheric pressure chemical ionization-MS

Triglycerides (TG) not only provide energy for infants but have important physiological functions. Understanding the composition and structure of TG in human milk is conducive to the development of infant formulas. In this study, TG species in human milk from 3 provincial capitals (Zhengzhou, Wuhan, and Harbin) in different regions of China were determined through C18 HPLC electrospray ionization tandem mass spectrometry (MS). The results showed that in human milk from these 3 regions, oleoyl-palmitoyl-linoleoylglycerol (OPL; 16.55, 19.20, and 18.67%, respectively) was more abundant than oleoyl-palmitoyl-oleoylglycerol (OPO; 10.08, 10.22, and 12.03%, respectively). Subsequently, regioisomeric and enantiomeric analysis of main TG in the human milk were performed on silver ion and chiral HPLC atmospheric pressure chemical ionization mass spectrometry (APCI)-MS, respectively. The results showed that rac-OPL (above 85%), rac-OPO (above 85%), rac-palmitoyl-oleoyl-oleoylglycerol (PPO; above 90%), and rac-OLaO (above 70%) were the main regioisomers of OPL, OPO, PPO, and lauroyl-oleoyl-oleoylglycerol (LaOO), respectively. The relative ratios of enantiomer pairs of rac-OPL (rac-OPL1 and rac-OPL2) were about 37 and 63%, respectively.

DETERMINATION OF CARBAMATE RESIDUES IN VEGATABLES, BULBS AND WATER

A method for the determination of residues of 10 carbamates including Aldicard Sulfoxide, Aldicarb sulfone, Oxamyl, Methomyl, 3-Hydroxycarbofuran, Aldicarb; Propoxur, Carbofuran, Carbaryl and Methiocarb in vegetables, bulbs and water was established based on High Performance Liquid Chromatography with fluorescence detection after derived post column. Solid samples were grinded in MeOH, clarify. Extraction liquid or water samples were cleaned by liquid-liquid extraction or SPE C18 Carbamates were extracted in HPLC C18 with mobile phase gradient MeOH/H2O and derived phthalaldehyde (OPA) and 2 - mercaptoethanol. A fluorescent compound 1 - hydroxytylthio - 2-metylisoindol was produced and detected by fluorescence detection with λex 340 nm and λem 445 nm. Limit of detection and limit of quantitation for the carbamates were 0.51 - 5.0 ppb and 1.69-9.09 ppb, respectively. The recoveries of the carbamates were from 73 to 95%. The concentrations of the carbamate in vegetables, bulbs and water at some investigated sites were low.

Forced degradation studies of norepinephrine and epinephrine from dental anesthetics: Development of stability‐indicating HPLC method and in silico toxicity evaluation

Injectable solutions containing epinephrine (EPI) and norepinephrine (NE) are not stable, and their degradation is favored mainly by the oxidation of catechol moiety. As studies of these drugs under forced degradation conditions are scarce, herein, we report the identification of their degradation products (DP) in anesthetic formulations by the development of stability-indicating HPLC method. Finally, the risk assessment of the major degradation products was evaluated using in silico toxicity approach. HPLC method was developed to obtain a higher selectivity allowing adequate elution for both drugs and their DPs. The optimized conditions were developed using a C18 HPLC column, sodium 1-octanesulfonate, and methanol (80:20, v/v) as mobile phase, with a flow rate of 1.5 mL/min, UV detection at 199 nm. The analysis of standard solutions with these modifications resulted in greater retention time for EPI and NE, which allow the separation of these drugs from their respective DPs. Then, five DPs were identified and analyzed by in silico studies. Most of the DPs showed important alerts as hepatotoxicity and mutagenicity. To the best of our acknowledgment, this is the first report of a stability-indicating HPLC method that can be used with formulations containing catecholamines.

Analysis of metoprolol enantiomers via reverse phase (RP-HPLC) with M-ß-Cyclodextrin as mobile additive

Enantiomeric separation of the racemic metoprolol was investigated using a reverse phase HPLC (RP-HPLC) with Methyl-beta-cyclodextrin (M-B-CD) as chiral mobile phase additive. A comparison of the enantioseparation of the racemic metoprolol, the system suitability, linearity, accuracy, limit of detection and quantification was undertaken to show the performance between two C18 columns, a Zorbax Eclipse XDB C-18 column (15 cm x 10 mm, 10 μm) and a Syncronis C18 HPLC column (250 mm x 4.6 mm, 5.0 μm) using high performance liquid chromatography with the same condition of mobile phase composition, pH value of the mobile phase and concentration of chiral additives. The resolution was achieved using a mobile phase consisting of a mixture of aqueous solution (3.5 g M-ß-CD in 300 ml H20), methanol with a volumetric ratio of 86:14 (v/v) and a flow rate of 1.0 ml/min and 0.5 ml/min for the columns respectively. Conversion of S-metoprolol found in this study are 60%, 51%, 15%.

Direct injection of pigment-protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC.

We discovered that pigments including carotenoids and (bacterio)chlorophylls in pigment-protein complexes, membrane fragments, and chlorosomes suspended in water could be injected directly into C18 HPLC and analyzed without any other treatments. We applied this method to LH1-RC and chromatophores of purple bacteria, chlorosomes of green sulfur bacteria, thylakoid membranes of cyanobacteria, and PSII and thylakoid membranes of spinach. HPLC elution profiles and pigment composition were the same as those of the conventional extraction method. The principle of this method might be that samples are first trapped on top of column, followed by the immediate extraction of the pigments with the HPLC eluent and their separation using the C18 column, as usual. In the conventional extraction method, pigments are first extracted with organic solvents, followed by evaporation of the solvents. The dried pigments are then dissolved in organic solvents and injected into C18 HPLC after filtration. The advantages of this method include the preventions of pigment isomerization and oxidation and the possibility of injecting all samples. Its drawbacks include the accumulation of denatured proteins at the top of column, causing increased HPLC pressure. The use of a guard column might solve this problem. Many factors, such as samples, column, and HPLC systems, may affect this method. Nevertheless, we think that some samples can be analyzed using this method.