Separation and Determination of the Structural Isomers of Madecassoside by HPLC Using β-Cyclodextrin as Mobile Phase Additive

A reversed phase HPLC method has been investigated for separation and determination of the structural isomers of madecassoside (madecassoside and asiaticoside-B). The isomeric compounds can be isolated with high resolution by adding β-CD in mobile phase on a C₁₈ column. The effect of β-CD concentration on resolution is discussed. The functional group in the separation process is investigated. The correlation coefficient (R ²) of the calibration was 0.9995 over the range of 0.1–5.0 mg mL⁻¹. The method was successfully applied to characterize and determine the madecassoside in Centella extract.     

Optimization of multicomponent solvent selection in high-performance liquid chromatography using a statistical method

A computer-assisted method is presented for optimization of multicomponent solvent mobile phase selection for separation of O-ethyl-N-isopropyl phosphoro (thioureido) thioates in reversed-phase HPLC and four geometric isomers of pesticides Decis in normal-phase HPLC. The method is based on Snyder's solvent selection triangle concept using a statistical method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted data and experimental results.     

Quantitative analysis of Cistanches Herba using high‐performance liquid chromatography coupled with diode array detection and high‐resolution mass spectrometry combined with chemometric methods

We aim to determine the chemical constituents of three species of Cistanches Herba using HPLC coupled with diode array detection and high‐resolution MS. Ten phenylethanoid glycosides were identified and further quantified as marker substances by HPLC coupled with diode array detection method. The separation was conducted using an Agilent TC‐C18 column with 0.1% formic acid and methanol as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of 36 batches of Cistanche plants. The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were used to compare different species of Cistanches Herba, leading to successful classification of the Cistanche samples in accordance with their origins. In conclusion, this study provides a chemical basis for quality control of Cistanches Herba.     

Determination of planar chlorobiphenyls in animal fat

The occurrence of the highly toxic non-ortho substituted (planar) chlorobiphenyls (CBs) at low levels in wildlife, (human) fat, and fish generates the need for a routine analytical procedure. The use of a fully automated system for the separation of planar and non-planar CBs, consisting of a gel permeation chromatography system, a sample preparation system employing extraction columns, and a porous graphitic carbon HPLC column is described, and tested with horse fat. Gas chromatography on capillary columns, with electron-capture or high resolution mass spectrometric detection, is used for quantitation of the CBs.     

Ultra high performance liquid chromatography with ion‐trap TOF‐MS for the fast characterization of flavonoids in Citrus bergamia juice

We have developed a fast ultra HPLC with ion‐trap TOF‐MS method for the analysis of flavonoids in Citrus bergamia juice. With respect to the typical methods for the analysis of these matrices based on conventional HPLC techniques, a tenfold faster separation was attained. The use of a core–shell particle column ensured high resolution within the fast analysis time of only 5 min. Unambiguous determination of flavonoid identity was obtained by the employment of a hybrid ion‐trap TOF mass spectrometer with high mass accuracy (average error 1.69 ppm). The system showed good retention time and peak area repeatability, with maximum RSD% values of 0.36 and 3.86, respectively, as well as good linearity (R² ≥ 0.99). Our results show that ultra HPLC can be a useful tool for ultra fast qualitative/quantitative analysis of flavonoid compounds in citrus fruit juices.     

Enantiomeric resolution,stereochemical assignment and toxicity evaluation of TPA enantiomers

Tetrahydro‐α ‐(1‐methylethyl)‐2‐oxo‐1(2H)‐pyrimidineacetic acid (TPA) is a critical intermediate in the synthesis of HIV protease inhibitors. A simple and efficient method for the separation and determination of TPA enantiomers was developed. The TPA was separated into its enantiomers with an enantiomeric purity of 99% using an HPLC system equipped with a Chiralpak OD‐H column. Semi‐preparative HPLC enantioseparations were carried out for further enrichment of the enantiomers. The validity of this method was evaluated on the basis of its precision, accuracy, linearity and recovery. The method was observed to be suitable for the rapid separation and semi‐preparation of TPA isomers. The separated enantiomers were identified by optical rotation and high‐resolution electrospray ionization mass spectrometry. Furthermore, the stereochemical structures of the TPA enantiomers were definitively confirmed using a combination of experimental and calculated electronic circular dichroism spectra. The toxicity of the separated pure enantiomers against Oryzias melastigma was evaluated using the median lethal concentration (LC₅₀) values. The results indicated that (S )‐(−)‐TPA is ~2.5 times more toxic than its enantiomorphism.     

TLC for pharmaceutical analysis in resource limited countries

This review article discusses the sustainability and robust advantages of planar chromatography that are critical to the successful performance of product quality assessments in resource limited areas including field applications. Because of the robustness and ease of use, the training required for successful performance of the high performance thin layer chromatography (HPTLC) assessments is much lower than that of other technologies with comparable reproducibility such as high performance liquid chromatography (HPLC). Some of the successful applications of planar chromatography in resource limited countries are presented. It should be noted that these planar chromatographic technologies have much lower plate counts and therefore separation power than column technologies such as HPLC and gas liquid chromatography (GLC). However in finished pharmaceutical products there are generally few active ingredients which are assessed making the HPTLC adequate for these analyses. In addition at this time there is a much wider array of detection technologies available for HPLC and GLC.     

Residues Analysis of Post-Harvest Imidazole Fungicides in Citrus Fruit by H PLC and GLC

Abstract

The determination of imazalil and prochloraz fungicide residues has been carried out by HPLC with an UV detector at 204 nm and by GLC with an electron capture detector (ECD).

In both cases fungicide residues were extracted with hexane/acetone (90:10, v/v) after pH adjustment and purified by a liquid-liquid partitioning process. When HPLC was used for prochloraz and imazalil analysis, it was necessary to eliminate the interfering substances with a further clean-up process. This was also required when samples with low residue levels were analyzed by GLC.

Recovery was always higher than 70%. The detection limit was 0.04 ppm for the HPLC method and 0.02 for the GLC method.

Imazalil and prochloraz residues in “Washington Navel” oranges and “Hernandina” clementine fruits, dipped in a 1000 ppm fungicide solution, are reported.     

Separation of Photosynthetic Pigments by High-performance Liquid Chromatography: Comparison of Column Performance,Mobile Phase,and Temperature

High-performance liquid chromatography (HPLC) has been commonly used as method of separating and identifying photosynthetic pigments such as chlorophylls and carotenoids because of such advantages as speed, high resolution and sensitivity. In this technique, high separation relies largely on the type of column material. This study compared the efficiency of five reverse-phase columns, C8, C18, C18 monolithic, π-NAP, and cholester, for separation of photosynthetic pigments at several fixed conditions of mobile phase and temperature. This investigation also analysed the parameters of Δt_R and t_R ratio for selected pigments and resolution for structural isomers, such as α- and β-carotene. Among above columns tested, cholester column is suitable for separation of pigments not only for a broad range of polarity, but also for hydrophobic pigments in a simple mobile phase. This finding can help in the selection of column and HPLC parameters in separating photosynthetic pigments.     

GLC and HPLC Determination of Diazepam and its Degradation Products in Pharmaceutical formulations

Abstract

Stability-indicating high performance liquid chromatographic (HPLC) and gas-liquid chromatographic (GLC) assays for diazepam in pharmaceutical formulations are described. In HPLC method, the material is extracted with 5 % aqueous methanol and chromatographed on a dimethyloctyl stationary phase using methanol-water-acetic acid (80: 20: 1) and propyl paraben internal standard. The system separated diazepam from the main degradation products, desmethyl diazepam (a synthetic precursor of diazepam) and the excipients present in ampoules and syrups. The GLC method included the extraction of diazepam and 2-methylamino-5-chiorobenzo-phenone (MACB) from aqueous acidic solution into chloro form leaving the other degradation products in the aqueous phase. The chloroform extract is evaporated to dryness, dissolved in chloroform containing diethylhexyl phtha-late as internal standard and chromatographed on an OV-17 stationary phase using flame ionisation detector. The results are compared with the BP method described for each formulation.     

Characterization of chemical constituents in Rhodiola Crenulate by high‐performance liquid chromatography coupled with Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS)

In this work, an approach using high‐performance liquid chromatography coupled with diode‐array detection and Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS) for the identification and profiling of chemical constituents in Rhodiola crenulata was developed for the first time. The chromatographic separation was achieved on an Inertsil ODS‐3 column (150 mm × 4.6 mm,3 µm) using a gradient elution program, and the detection was performed on a Bruker Solarix 7.0 T mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 48 chemical compounds, including 26 alcohols and their glycosides, 12 flavonoids and their glycosides, 5 flavanols and gallic acid derivatives, 4 organic acids and 1 cyanogenic glycoside were identified or tentatively characterized. The results indicated that the developed HPLC‐FT‐ICR MS method with ultra‐high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents in R. crenulata. And it provides a helpful chemical basis for further research on R. crenulata. Copyright © 2016 John Wiley & Sons, Ltd.     

Oil-in-Water Microemulsion High Performance Liquid Chromatographic Analysis of Pharmaceuticals

Efficient and novel oil-in-water microemulsion HPLC separations of a range of solutes have been achieved using isocratic elution and a conventional reversed-phase HPLC column. A single set of isocratic oil-in-water microemulsion HPLC conditions are shown to be successful for the resolution of a wide range of basic, neutral and acidic drugs and excipients. A robust separation method was developed for the quantitative analysis of Naproxen in a tablet formulation. The method offered a similar run time and improved chromatography when compared to conventional HPLC modes, demonstrating its potential for routine use. In developing the separations the effect on the chromatography of varying the operating parameters was studied. While generally the method was robust to changes in some variables, others markedly changed the separation selectivity, solute retention, peak-peak resolution or resulted in poor chromatographic performance. The method was found to be compatible with very low UV working detection wavelengths. As a final stage to the experimental programme, a microemulsion method was developed with the capability of analysing a range of water-insoluble drugs.     

Gas Chromatographic Separation of Enantiomers on Optically Active Metal-Complex-Free Stationary Phases. New Analytical Methods (24)

If a stationary phase A employed in gas chromatography possesses a chemical affinity for substance B, which is to be separated, then the retention behavior is not only determined by the normal physical equilibrium between the gas and liquid phases but also by the chemical equilibrium A + B ? AB. If A and B are chiral and A is present in optically active form while B is a racemic mixture, then it is possible to achieve a gas chromatographic enantiomer resolution without the isolation of diastereomers: the energetically different diastereomeric associates A_R B_R and A_R B_S are formed rapidly and reversibly. This enantiospecific resolution principle was first demonstrated in 1966 by the quantitative resolution of racemic amino acid derivatives on optically active peptide phases in analogy to the well-known stereospecificity of enzymes. The anchoring of the chiral resolving agent to thermally stable polysiloxanes together with the employment of high resolution capillary columns and the use of appropriate derivatization strategies has led to the development of enantiomer resolution into a routine modern method for many classes of substances. The demonstration of enantiospecificity in the gas chromatographic separation process is of fundamental interest, and its systematic study can result in a significant contribution to the understanding of the molecular mechanism of “chiral recognition”. The gas chromatographic separation of enantiomers has also proven to be an accurate and sensitive method for the determination of the enantiomeric composition of natural products and products of enantioselective transformations (asymmetric syntheses, “chiral pool” transformations, kinetic resolutions, biomimetic reactions) and for the quantification of racemization, e.g. in the synthesis and hydrolysis of peptides. In any research program devoted to the phenomenon of chirality, the gas chromatographic separation of the enantiomers of volatile compounds constitutes an indispensable modern instrumental technique.     

High‐resolution separation of homogeneous chitooligomers series from 2‐mers to 7‐mers by ion‐exchange chromatography

Highly purified chitooligomers with single degree of polymerization are of significance for studying bioactivity of chitooligomers. However, there are few reports on high‐resolution preparative separation of chitooligomers, especially for those oligomers with degree of polymerization higher than 4. This study developed a high‐resolution chromatography for the preparative separation of a pure fully deacetylated chitooligomer series. A glucosamine oligomer mixture with low degree of polymerization was prepared by acid hydrolysis of a highly deacetylated chitosan. Then, six fractions were separated from the prepared oligomer mixture by ion‐exchange chromatography and analyzed by HPLC and ESI/MS, which primarily contained glucosamine dimers, trimers, tetramers, pentamers, hexamers, and heptamers, respectively, with chromatographic purities over 98% for dimers to hexamers and a purity of 93% for heptamers. The yields of a single round of separation were 75, 60, 60, 55, 35, and 20 mg for glucosamine dimers, trimers, tetramers, pentamers, hexamers, and heptamers, respectively. Furthermore, a chromatographic separation model for GlcN homomers was established. The capacity factor (k) of glucosamine oligomers and their degrees of polymerization (DPs) exhibited a good correlation, lnk = 0.786 + 0.846 lnDP, (R² = 0.997). Based on this equation, glucosamine octamers are expected to be separated by this system.     

Performance of HPLC/MS microchips in isocratic and gradient elution modes

We analyzed the chromatographic performance of particle‐packed, all‐polyimide high‐performance liquid chromatography/mass spectrometry (HPLC/MS) microchips in terms of their hydraulic permeabilities and separation efficiency under isocratic and gradient elution conditions. The separation channels of the chips (with ca 50 µm × 75 µm trapezoidal cross‐section and a length of 43 mm) were slurry packed with either 3.5 or 5 µm spherical porous C18‐silica particles. A custom‐built holder enveloped the chip during packing to prevent channel deformation and delamination from high pressures. It is shown that the packing conditions significantly impact the packing density of the HPLC/MS chips, which determines their performance in both, isocratic and gradient elution modes. Even with steep solvent gradients, peak shape and chromatographic resolution for the densely packed HPLC/MS chips are much improved. Our data show that the analytical power of the HPLC/MS chip is limited by the quality of the chromatographic separation. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of Iso-Selective Gradient Elution for the Separation of Selected Phthalates

Abstract

In this paper, the overlapping resolution mapping scheme was applied to the gradient HPLC separation of selected phthalates. The procedure employs a minimum of seven experiments for the determination of the optimum mobile phase gradient for the desired separation. In the present work, solvent systems for iso-selective multisolvent gradient elution were used. The overlapping resolution mapping procedure was found to be a rapid and systematic approach for optimizing HPLC separations.     

Oil-In-Water Microemulsion LC Determination of Pharmaceuticals Using Gradient Elution

Efficient and novel oil-in-water microemulsion HPLC (MELC) separations of a range of solutes have been performed on conventional reversed-phase HPLC columns using gradient elution. This work follows previous successful separations using isocratic oil-in-water MELC [1]. It was found that by changing certain variables, peak-peak resolution, separation selectivity, efficiency and solute retention could be manipulated. The method was compatible with very low UV detection wavelengths. A robust separation method was developed for the quantitative analysis of 2 steroids in a combination-inhaled product for asthma. The method offered similar chromatography and run time when compared with conventional HPLC modes, thus demonstrating its potential for routine use. Stability-indicating methods were developed to separate synthetic and degradative impurities from the main component peaks in 4 pharmaceutical products. The methods offered quicker analysis times and equivalent selectivity to conventional HPLC modes. In developing the separations the effect on the chromatography of varying the operating parameters was studied.     

Temperature-dependent separation of bryostatin 18 and 10 by high performance liquid chromatography

Summary The temperature-dependent separation of bryostatins by HPLC was examined on an octadecyl bonded stationary phase, using column temperatures between 0 and 40°C and mobile phase temperatures from 0 to 25°C. The retention time and resolution of bryostatins changed drastically and separation improved with decreasing temperature. A column temperature of less than 5°C and a mobile phase temperature of less than 15°C is recommended for a good resolution of bryostatins for routine work.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.     

UHPLC: The Greening Face of Liquid Chromatography

Pharmaceutical analysis based on chromatographic separation is an important part of studies aimed at developing routine quality analysis of drugs. High-performance liquid chromatography (HPLC) is one of the main analytical techniques recommended for drug analysis. Although it meets many criteria vital for analysis, it is time-consuming and uses a relatively high amount of organic solvents compared to other analytical techniques. Recently, Ultra-high-performance liquid chromatography (UHPLC) has been frequently proposed as an alternative to HPLC, which means introducing an environment-friendly approach to drug analysis achieved by reducing the consumption of solvents. It also offers greater chromatographic resolution and higher sensitivity as well as requiring less time due to faster analysis. This review focuses on the basics of UHPLC, compares that technique with HPLC and discusses the possibilities of applying UHPLC for the analysis of different pharmaceuticals and biopharmaceuticals.