Short HPLC Column Research Articles

Study of Mono and Di-O-caffeoylquinic Acid Isomers in Acmella oleracea Extracts by HPLC-MS/MS and Application of Linear Equation of Deconvolution Analysis Algorithm for Their Characterization.

Chlorogenic acids, the esters of caffeic and quinic acids, are the main phenolic acids detected in Acmella oleracea extracts and have gained increasing interest in recent years due to their important biological activities. Given their structural similarity and instability, the correct analysis and identification of these compounds in plants is challenging. This study aimed to propose a simple and rapid determination of the A. oleracea caffeoylquinic isomers, applying an HPLC-MS/MS method supported by a mathematical algorithm (Linear Equation of Deconvolution Analysis (LEDA)). The three mono- and the three di-caffeoylquinic acids in roots of Acmella plants were studied by an ion trap MS analyzer. A separation by a conventional chromatographic method was firstly performed and an MS/MS characterization by energetic dimension of collision-induced dissociation mechanism was carried out. The analyses were then replicated using a short HPLC column and a fast elution gradient (ten minutes). Each acquired MS/MS data were processed by LEDA algorithm which allowed to assign a relative abundance in the reference ion signal to each isomer present. Quantitative results showed no significant differences between the two chromatographic systems proposed, proving that the use of LEDA algorithm allowed the distinction of the six isomers in a quarter of the time.

Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC)

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic.The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

Preparation of terbutylazine imprinted polymer microspheres using viscous polymerization solvents

Spherical micron sized terbutylazine imprinted polymers have been obtained by copolymerization of acrylates in solvent mixtures containing a highly viscous ('oil') component. The new method requires much less organic solvent than precipitation polymerization to produce spherical MIP particles. Different proportions of oil have been used to clarify its role on the morphology. The particles obtained with the optimal composition could be easily packed into short HPLC columns. Chromatographic retention of the template and other compounds has been determined on the novel ('OMIP') columns and was compared to MIP prepared by bulk polymerization. In 70% aqueous acetonitrile eluent the OMIP shows a higher imprinting factor (i. e. less non-selective binding) and lower retention than the bulk MIP. The chromatographic selectivity against template analogs is similar on OMIP and bulk MIP, while the selectivity against non-related substances is better on the OMIP. The effect of oil on the polymer structure appears to be due to its high viscosity.

Method of analysis of a selected group of pyrethroids in soil samples using off-line flow-through extraction and on-column direct large-volume injection in reversed phase high performance liquid chromatography

An analytical method using flow-through extraction of a soil sample filled in a short HPLC column with methanol or methanol-water mixtures and large-volume injection in RP-HPLC has been developed for the simultaneous determination of residues of three pyrethroids--kadethrin, cypermethrin, and permethrin--from soil samples. The developed RP-HPLC method enables separation of four diastereoisomers of cypermethrin into three peaks and resolution of two diastereoisomers of permethrin. The UV photometric detection limits of direct on-column large-volume injection of 1.00 mL of extract were 30 ng/mL of kadethrin, 37 ng/mL of total content of cypermethrin, and 65 ng/mL of trans-permethrin, which corresponds to a pyrethroid soil content of around 0.3 mg/kg. Effects of extractant flow rate and optimal extractant volume on the percentage recovery of pyrethroids from Slovak soil samples were studied. Recovery studies were performed at 0.5- 5.0 microg/g fortification level of kadethrin and 1.0-2.5 microg/g fortification level of cypermethrin and permethrin in a soil sample. Recoveries ranged from 83 to 90% for kadethrin, from 87 to 94% for total cypermethrin, and from 85 to 98% for trans-permethrin. This work comprises a basic study aimed at elaboration of an RP-HPLC method of direct analysis of pyrethroids in a soil matrix at low concentration levels achieved by a "solid sample injection" in HPLC--on-line interfacing of analyte extraction, extract clean-up, and analysis.

Influence of the TiO 2 content on the chromatographic performance and high pH stability of C 18 titanized phases

To extend pH stability, protective metal oxide layers, such as titanium oxide, that are more stable in alkaline medium, can be chemically bonded to the chromatographic silica surface prior to reaction with silanes. In the present work, the influence of the titanium oxide content on the chromatographic performance was investigated by synthesizing a C 18 phase onto a doubly-titanized silica support and comparing its chromatographic performance with a C 18 phase on singly-titanized silica. The Engelhardt and Tanaka test mixtures were used for chromatographic characterizations using short HPLC columns. The column lifetimes of these titanized phases were also compared by performing accelerated aging tests at 50 °C using aggressive phosphate mobile phases at pH 10.

Simultaneous determination of four immunosuppressants by means of high speed and robust on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry

In this study immunosuppressants, i.e. cyclosporin A (CyA), tacrolimus (TRL), sirolimus (SRL) and everolimus (RAD) were quantified in whole blood samples from immunosuppressant treated transplant recipients by an integrated on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry (SPE–HPLC–MS/MS) system. This method has been developed to improve the following characteristics: speed, robust analysis, simultaneous determination and low cost. This can be achieved by the use of a perfusion column as an extraction cartridge in combination with a short HPLC column and highly selective and sensitive atmospheric pressure ionisation tandem mass spectrometry (API–MS/MS) in the multiple reaction monitoring (MRM) detection mode. This high throughput technique is perfectly appropriate for routine therapeutic drug monitoring (TDM) of organ transplanted patients.

In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry.

Metabolite identification can provide critical information for further synthesis during the lead optimization stage of drug discovery. Fast chromatographic gradient methodology was explored to reduce the analysis time for metabolite identification by liquid chromatography/tandem mass spectrometry while maintaining chromatographic separation of all components of interest. Short HPLC columns (2 cm) containing 3 microm particles were used and the chromatographic conditions involved elevated flow rates (1.0-2.5 mL/min) and fast, full range linear gradients (<2 min). Detection was accomplished by fast scanning triple quadrupole mass spectrometers or a quadrupole time-of-flight mass spectrometer. Analysis of liver microsomal incubations of several compounds indicates that separation of isomeric metabolites as well as the parent compound is feasible with analysis times not exceeding 2 minutes.

Ultra-fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods developed for quantification using rapid ('ballistic') gradients on narrow bore, short HPLC columns have been previously described by this laboratory. This paper compares the fast gradient approach with the more traditional high-organic isocratic LC/MS/MS methods. The comparison is based on an analysis of the effectiveness of the chromatographic separations when using the two approaches (i.e. k', N, and W). The data presented herein are derived from actual biological samples analyzed as part of the drug discovery process.

Advantages of the Use of Very Short and Ultra Short Hplc Columns for Drug Analysis in Dissolution Testing

The advantages of short (3–5cm long) or ultra short (4mm long) HPLC columns for drug analysis in dissolution testing are illustrated by reference to some example antihypertensive drug formulations.Advantages include: selectivity, where interfering excipients or co-formulated drugs complicate UV spectrophotometric analysis: time savings, compared with conventional HPLC columns (although where UV spectrophotometry is applicable no time advantages may be obtained with short column HPLC): economy, as the columns are less expensive than conventional columns and there is reduced solvent consumption: increased sensitivity compared with conventional columns. Low dose potent. drugs with poor chromophores may be more readily quantitated: amenity to automation, including the use of laboratory robots.These advantages would suggest the wide applicability to this mode of analysis in dissolution testing.