Gradient High-performance Liquid Chromatography Research Articles

Determination of Rosmarinic Acid by High-Performance Liquid Chromatography and Its Application to Certain Salvia Species and Rosemary

This study describes a method development for the determination of rosmarinic acid (RA) by using a gradient high-performance liquid chromatography (HPLC) and its application to certain plant materials. The analysis was performed by utilizing a two solvents system [A: methanol/water/formic acid (10:88:2; v:v:v); B: methanol/water/formic acid (90:8:2; v:v:v)] on a reverse-phase column. The flow rate and injection volume were 1 ml min−1 and 10 μl, respectively. Signals were detected at 280 nm. In addition, an internal standard (IS) technique was applied for the analysis of RA to increase precision, and propylparaben was employed for this purpose. The repeatability results as RSD% were 1.66, 1.17 and 1.26 for intra-day and 1.38 was for inter-day with the employment of (3.67 × 10−5 M) RA. A limit of linearity (LOL) was observed in a wide (1.13 × 10−5–5.65 × 10−4 M) concentration range. Linearity parameters were also examined in the range of 5.95 × 10−6–7.14 × 10−5 M RA, and very good correlation was observed. The limit of detection (LOD) and limit of quantification (LOQ) (for inter-day) were 1.60 × 10−6 M (signal/noise [S/N] = 3.3) and 4.80 × 10−6 M (S/N = 10), respectively. The method was applied to the extracts of certain Lamiaceae plants (Salvia candidissima Vahl. subsp. candidissima, S. sclarea L., S. verticillata L. subsp. verticillata and R. officinalis L.), and reasonable results were obtained.

Developing methods for analysis of the solid dosage form (capsules) of cardiocyclide

We have developed methods for the pharmaceutical analysis of the new domestic antiarrhythmic drug cardiocyclide in a solid dosage form (capsules). The main pharmacopoeial quality tests were carried out. The content of impurities in capsules was determined by thin-layer chromatography. UV spectrophotometry and gradient high-performance liquid chromatography were used for simultaneous assay, purity check, and quantitative analysis of the drug. The pharmacopoeial “Dissolution” test was carried out using UV spectrophotometry.

Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

The purpose of this work was to test the applicability of the current theory to predict the peak retention time and the peak width in the combined pH/organic modifier gradient reversed phase high performance liquid chromatography (RP HPLC). A series of 38 isocratic measurements have been conducted for a wide range of pH and methanol contents for ketoprofen (weak acid) and papaverine (weak base). It served to find the model describing dependence of retention factor and the height equivalent of a theoretical plate (HETP) on pH and organic modifier content. The information gathered in the isocratic mode was used to simulate retention times and peak widths for 30 various methanol gradients, 25 pH gradients, and 3 combined pH/methanol gradients. The simulations were compared with the experimental data. We also proposed a simplified version of this model that was parameterized based on 12 initial organic modifier gradients carried out for different pHs and for the 20 min and 60 min gradient development times. The full and the simplified model described the experimental data very well. In conclusion, the proposed modeling approach allowed predicting analyte retention times and peak width for various pH and organic modifier changes. Its simplified version required only 12 initial experiments and seems to be very promising in the optimization RP HPLC separations for complex samples and for conditions providing peak compression.

Trace analysis of endectocides in milk by high performance liquid chromatography with fluorescence detection

An analytical method has been developed for the simultaneous determination of the following endectocide drugs in milk: ivermectin, abamectin, doramectin, moxidectin, eprinomectin, emamectin and nemadectin. Samples were extracted with acetonitrile, purified with solid-phase extraction on a reversed phase C 8, derivatised with N-methylimidazole, trifluoroacetic anhydride and acetic acid to a stable fluorescent derivative, and were further analysed by gradient high performance liquid chromatography (HPLC) on an endcapped reversed phase Supelcosil LC-8-DB. The derivatisation step was mathematically optimised and the method was validated according to the requirements of Commission Decision 2002/657/EC, using fortified raw bovine milk. Mean recovery was between 78 and 98%. The repeatability (CV r) and within-laboratory reproducibility (CV W) ranged from 4.6 to 13.4% and from 6.6 to 14.5%, respectively. Decision limits (CCα) for analytes with MRL values, namely eprinomectin and moxidectin, were determined to be 24.8 and 50.6 μg kg −1, respectively. CCα values for unauthorised endectocides ranged from 0.1 to 0.2 μg kg −1. Due to high acceptability regarding the required criteria and applicability to ovine and caprine milk, giving similar results, this multi-analyte method has been successfully implemented in pharmacokinetic research studies as well as statutory residue monitoring in Slovenia.

Development and Validation of a Novel Gradient LC Method for Simultaneous Determination of Isoniazid and Acetylisoniazid in Human Plasma

The goal of this study was to develop and validate a new gradient high-performance liquid chromatography method for the simultaneous determination of isoniazid (INH) and acetylisoniazid (Ac-INH) in human plasma samples. A C18 reversed-phase column was employed for separation followed by UV detection at 266 nm. The calibration involved the use of five concentration levels ranging from 1 to 20 μg mL−1 for both analytes. The developed method was validated using ICH guidelines. The calibration curve was found to be linear with correlation coefficient values (r2) above 0.9991 and the highest RSD% values for intra-day assays were found to be 6.34 and 2.57% for INH and Ac-INH, respectively. The highest RSD% values for inter-day assays were 9.31 and 10.17% for INH and Ac-INH, respectively. LOD was calculated to be 0.1 and 0.15 μg mL−1 for INH and Ac-INH, respectively. LOQ was calculated to be 0.33 and 0.5 μg mL−1 for INH and Ac-INH, respectively.

Procedure for Automated Background Correction in Flow Systems with Infrared Spectroscopic Detection and Changing Liquid-Phase Composition

This paper describes a partial least squares (PLS) based automatic procedure to correct for changes in the spectral contribution of the solvent or solvent mixtures from solute spectra recorded in these solvents. The procedure was developed for successful on-line Fourier transform infrared (FT-IR) detection in gradient high-performance liquid chromatography (HPLC) separations. It requires a reference FT-IR data set containing all possible combinations of the expected variation in solvent composition. Furthermore, a spectral region (A) in these spectra is required where the solvents show absorption but the analytes do not. This is the case for the system ACN:H(2)O, an often-applied solvent mixture in gradient HPLC, in the spectral region from 2300-2400 cm(-1). By focusing on (A) the developed numerical method selects an appropriate background spectrum from the reference data set, which is then subtracted from the analyte spectra. The method was programmed in Matlab and tested on different isocratic and gradient on-line reversed phase liquid chromatography-Fourier transform infrared spectrometry (LC-FT-IR) data sets. This work describes a successful method to perform eluent correction in on-line coupling of FT-IR spectrometry with gradient LC.

Rapid optimization of dual-mode gradient high performance liquid chromatographic separation of Radix et Rhizoma Salviae Miltiorrhizae by response surface methodology

An approach for rapid optimization of dual-mode gradient high performance liquid chromatography (HPLC) by response surface methodology (RSM) was developed for fast simultaneous separation of hydrophilic and hydrophobic components in Radix et Rhizoma Salviae Miltiorrhizae (Danshen) and its preparations. The aim of this study was to achieve a high throughput RSM optimization using a short ultra-high performance liquid chromatographic (UHPLC) column to simultaneously optimize flow rate and solvent gradient, and then transfer the optimized method to conventional HPLC for routine analytical purposes. The optimization was designed with Box Behnken design (BBD) and the global Derringer's desirability was used for describing the multicriteria response variables. Sixty-two designed experiments were performed by UHPLC with a short sub-2 μm column (2.1 mm × 50 mm, 1.7 μm) and a total running time of only 5 h. The predicted gradient profile was further transferred to a long UHPLC column (2.1 mm × 100 mm, 1.7 μm) and a conventional HPLC columns (2.1 mm × 100 mm, 3.5 μm and 4 mm × 100 mm, 5 μm, respectively). Compared to the published methods, the newly developed dual-mode gradient is faster and more efficient at simultaneously separating hydrophilic and hydrophobic components in Danshen and its preparations.

Evaluation of Methods for the Simultaneous Analysis of Cations and Anions Using HPLC with Charged Aerosol Detection and a Zwitterionic Stationary Phase

This paper describes the development and qualification of a method capable of analyzing inorganic ions as salts and counter-ions of both active pharmaceutical ingredients and other compounds such as lysine. The use of a polymeric zwitterionic column with a binary high-performance liquid chromatography gradient enables the separation of several anions and cations in a single run. A generic gradient (method #1) was developed and validated with respect to specificity, correlation, intermediate precision, accuracy, and sensitivity (limits of quantitation and detection) for four anions and two cations. Furthermore, the ability to alter chromatographic selectivity by simple gradient manipulation (without altering the mobile phase composition or column type) is demonstrated for nine anions and three cations (method #2). The simultaneous measurement of cations and anions at the parts per billion level using the Corona charged aerosol detector with zwitterionic chromatography-polymeric hydrophilic interaction chromatography is a viable alternative to traditional techniques used for ion analysis.

Analysis of Nootsetam syrup by gradient high-performance liquid chromatography

An original procedure was suggested for analyzing a new multicomponent medicinal preparation, Nootsetam syrup, by high-performance liquid chromatography in a linear gradient mode. The confidence of results was confirmed upon the analysis of model solutions containing active main, preservatives, and all auxiliary components. Analysis of tentative samples of the syrup was performed, all results correspond to specifications. The protocol of the analysis is included in the factory standard for this pharmaceutical preparation.

Identification and characterization of potential impurities of valsartan, AT1 receptor antagonist

Five impurities (related substances) were detected during the impurity profile study of an antihypertensive drug substance, valsartan. A simple gradient high performance liquid chromatographic method (HPLC) and liquid chromatography–mass spectrometry (LC–MS) were used for the detection. Based on the spectral data (IR, NMR and MS) followed by synthesis, these impurities were characterized as ( S)- N-(1-carboxy-2-methylprop-1-yl)- N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity I); ( S)- N-(1-carboxy-2-methylprop-1-yl)- N-(5-phenylthio)pentanoyl- N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity II); ( S)- N-(1-carboxy-2-methylprop-1-yl)- N-(5-phenyl)pentanoyl- N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity III); ( S)- N-(1-carboxy-2-methylprop-1-yl)- N-4-pentenoyl- N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity IV); ( S)- N-(1-carboxy-2-methylprop-1-yl)- N-(5-hydroxy)pentanoyl- N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity V).

Identification, characterization and synthesis of impurities of zafirlukast

Zafirlukast is a drug in the treatment of pulmonary disorders such as asthma. During the process development of zafirlukast, five unknown impurities were detected at levels of below 0.10% (ranging from 0.05 to 0.15%) in reverse phase gradient high performance liquid chromatography (HPLC) method. The molecular weights were determined by LC–MS analysis. These impurities were isolated from crude samples of zafirlukast using gradient reverse phase preparative HPLC and were subsequently synthesized. Based on the spectral data, the structures of these impurities were characterized as 3-methoxy-4-(5-methoxycarbonylamino-1-methyl-1 H-indol-3-ylmethyl)-benzoic acid (Impurity 1), {3-[2-methoxy-4-(toluene-2-sulfonylaminocarbonyl)-benzyl]-1-methyl-1 H-indol-5-yl}-carbamic acid methyl ester (Impurity 2), {3-[2-methoxy-4-(toluene-3-sulfonylaminocarbonyl)-benzyl]-1-methyl-1 H-indol-5-yl}-acetic acid cyclopentyl ester (Impurity 3), {3-[2-methoxy-4-(toluene-4-sulfonylaminocarbonyl)-benzyl]-1-methyl-1 H-indol-5-yl}-acetic acid cyclopentyl ester (Impurity 4), and 4-(5-cyclopentyloxy carbonylamino-1-methyl-1 H-indol-3-yl methyl)-3-methoxy-benzoic acid methyl ester (Impurity 5). The separation of the impurities by reverse phase HPLC, the confirmation of their structures by IR, MS and NMR spectral data, the mechanism of their formation and their syntheses are discussed in detail.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Application of gradient high-performance liquid chromatography to the analysis of some multicomponent pharmaceutical preparations

Procedures were developed for determining streptocide and norsulfazole in the Ingalipt aerosol; dexpanthenol and nipagin in the Dekspantenol aerosol; and oxymetazoline and benzalkonium chloride in a nasal spray by reversed-phase high-performance liquid chromatography (HPLC) in the gradient elution mode using the CH3CN-0.025 M aqueous solution mixture of KH2PO4 (pH 3.0) and UV detection. The developed procedures are comparable in accuracy and precision to chromatographic procedures recommended by regulatory documents; however, they ensure the simultaneous determination of all components of the pharmaceutical preparations and are more rapid and less laborious.

Recovery and long-term renal excretion of propofol, its glucuronide, and two di-isopropylquinol glucuronides after propofol infusion during surgery

BackgroundThe metabolism of the short-acting anaesthetic agent propofol has been described over the first 24 h. However, the long-term disposition of propofol and its metabolites is unclear. We describe the pharmacokinetics (renal excretion rates and renal clearance) of propofol and its metabolites over 60 h. MethodsTen patients undergoing lung surgery were included in the study. They received anaesthesia with continuous i.v. propofol at an average rate of 10 mg min−1. During surgery and 60 h thereafter, we sampled blood and urine. Propofol and its metabolites were measured using gradient high performance liquid chromatography (HPLC). ResultsIn nine patients, propofol and its glucuronides were found in the plasma over the first 15 h. In the urine, however, even after 60 h, propofol and its quinol glucuronides were still detectable. One patient had a markedly different pharmacokinetic profile, showing a limited renal excretion or absorption of 12% of the dose. ConclusionsAfter an infusion of propofol, patients excrete propofol and its metabolites in the urine over a period in excess of 60 h. We hypothesize that (re)absorption of propofol and its metabolites by the kidney is a major process in elimination and that the reabsorbed compounds are gradually conjugated in the kidney and excreted in the urine. One patient showed a different pharmacokinetic profile for which we currently have no explanation.

Evaluation of the stability of morphine sulphate in combination with Instillagel®

The Pharmacy Department at the Western General Hospital in Edinburgh prepares a topically applied product in which morphine sulphate is incorporated into Instillagel for use in reducing pain associated with rectal and other cancers. A stability indicating method was required for assessment of the stability of this combination. A gradient high performance liquid chromatography method was developed to assess stability and an LC-MS method was used to characterize the degradants from forced degradation of the components in the formulation. The method was found to have acceptable inter- and intra-day precisions. Under all storage conditions investigated morphine sulphate remained within 98.1% and 103.2% of the initial concentration, lidocaine hydrochloride within 96.07% and 104.9% and chlorhexidine gluconate within 97.3% and 105.5%, except for samples stored at 37 degrees C beyond 240 h. A sample of the admixture was stored up to 7 months at 37 degrees C and was found to be reasonably stable with only the chlorhexidine concentration declining appreciably to 92% of the initial concentration. Some of the degradants of chlorhexidine and morphine were characterized by liquid chromatography mass spectrometry. It could be concluded that the admixture was stable for over 22 days at 4 degrees C protected from light, over 22 days at 25 degrees C exposed to normal light, and for 7 months at 37 degrees C protected from light. The components in the combination were found to have good long-term stability.

Effect of column loading on the accuracy of analyzing pentalgin N tablets by gradient high-performance liquid chromatography

Two approaches for the high-performance liquid chromatographic analysis of multicomponent medical preparations were experimentally studied by an example of Pentalgin N tablets. One of them was the simultaneous determination of all components of the preparation; another was the independent analysis of concentrated and diluted solutions for determining the presentpresent in small and large amounts, respectively. The performance characteristics of the studied procedures were determined in the analysis of model solutions containing all active and adjuvant substances of the tablets. In the simultaneous determination of all components, the relative error was smaller because of the more sustained performance of the chromatographic column in the absence of its overload with the sample observed in the independent analysis.

Simultaneous analysis of different classes of phytohormones in coconut ( Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry after solid-phase extraction

Coconut ( Cocos nucifera L.) water, which contains many uncharacterized phytohormones is extensively used as a growth promoting supplement in plant tissue culture. In this paper, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of various classes phytohormones, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N 6-benzyladenine (BA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid, pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detection made at 265 nm wavelength. The method was validated for specificity, quantification, accuracy and precision. After preconcentration of putative endogenous phytohormones in CW using C 18 solid-phase extraction (SPE) cartridges, the HPLC method was able to screen for putative endogenous phytohormones present in CW. Finally, the identities of the putative phytohormones present in CW were further confirmed using independent liquid chromatography–tandem mass spectrometry (LC–MS/MS) equipped with an electrospray ionization (ESI) interface.

Gradient HPLC analysis of raloxifene hydrochloride and its application to drug quality control

A rapid, sensitive and selective method for the determination of raloxifene hydrochloride (RLX) in pure drug and in tablets was developed using gradient high performance liquid chromatography (HPLC). The devised method involved separation of RLX on a reversed phase Hypersil ODS column and determination with UV detection at 284 nm. The standard curve was linear (R = 0.999) over the concentration range of 50-600 microg mL-1 with a detection limit of 0.04 microg mL-1 and a quantification limit of 0.16 microg mL-1. Intra-day and inter-day precision and accuracy of the method were established according to the current ICH guidelines. Intra-day RSD values at three QC levels (250, 450 and 550 microg mL-1) were 0.2-0.5%, based on the peak area. The intra-day relative error (er) was between 0.2 and 0.5%. The developed method was successfully applied to the determination of RLX in tablets and the results were statistically compared with those obtained by a literature method. Accuracy, evaluated by means of the spike recovery method, was the excellent with percent recovery in the range 97.7-103.2 with precision in the range 1.6-2.2%. No interference was observed from the co-formulated substances. The method was economical in terms of the time taken and the amount of solvent used.

High-temperature gradient HPLC and LC-NMR for the analysis of complex polyolefins

Abstract The synthesis and characterization of polyolefins continues to be one of the most important areas for academic and industrial polymer research. One consequence of the development of new "tailor-made" polyolefins is the need for new and improved analytical techniques for the analysis of polyolefins with respect to molar mass and chemical composition distribution. The present article briefly reviews different new and relevant techniques for polyolefin analysis. Crystallization analysis fractionation is a powerful new technique for the analysis of short-chain branching in linear low-density polyethylene (LLDPE) and the analysis of polyolefin blends and copolymers regarding chemical composition. For the fast analysis of the chemical composition distribution, a new high-temperature gradient high-performance liquid chromatography (HPLC) system has been developed. The efficiency of this system for the separation of various olefin copolymers is demonstrated. The correlation between molar mass and chemical composition can be accessed by on-line coupling of high-temperature size exclusion chromatography (HT-SEC) and 1H NMR spectroscopy. It is shown that the on-line NMR analysis of chromatographic fractions yields information on microstructure and tacticity in addition to molar mass and copolymer composition.

New gradient high-performance liquid chromatography method for determination of donepezil hydrochloride assay and impurities content in oral pharmaceutical formulation

A new gradient HPLC method has been developed and validated for the determination of both assay and related substances of donepezil hydrochloride in oral pharmaceutical formulation. Different kinds of columns and gradient elution programs were tested in order to achieve satisfactory separation between the active substance, four impurities and an interfering excipient used in the formulation. The best results were obtained using an Uptisphere ODB C-18 column 250 mm × 4.6 mm, 5 μm, UV detection at 270 nm and a gradient elution of phosphate buffer (0.005 M, pH 3.67) and methanol as the mobile phase. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. It was also found to be stability indicating, and therefore suitable for the routine analysis of donepezil hydrochloride and related substances in the pharmaceutical formulation.