Biomedical subjects

Torgny Fornstedt

Publications and source records attributed to Torgny Fornstedt.

13 recordsLinked to original sources

PubMed2006

General theory of indirect detection in chromatography.

Indirect detection is used in liquid chromatography to detect sample components that more or less lack detectable properties. A detectable component, the probe, is added to the mobile phase and the sample containing the components is injected. Every solute, even those not normally detectable, will give rise to detectable peaks due to the presence of the probe. The theory currently used to describe this situation is different for different adsorption isotherm models and also restrictions have to be imposed on the adsorption isotherm parameters. We present a general theory that describes both the retention times and areas of the detected peaks without making any a priori adsorption isotherm assumptions. Our general theory is compared with the current theory and validated by computer simulations. An alternative quantification method is suggested based on the measurement of the relative areas of the sample components without the requirement of having standard curves of known solutions of the sample components. Using this approach, the new theory is able to predict the solute concentrations in cases when the current theory did not, i.e., when the saturation capacities of the sample components and the probe differed from each other. In addition, a new application is proposed: the estimation of adsorption isotherm parameters.

Adsorption↗

PubMed2006

Validation of the tracer-pulse method for multicomponent liquid chromatography, a classical paradox revisited.

The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.

Journal Article↗

PubMed2006

Invisible analyte peak deformations in single-component liquid chromatography.

It is well known that if a small excess of solute is injected into a chromatographic system equilibrated with an eluent containing the same solute, a single so-called perturbation peak will appear in the chromatogram. It was recently shown (Samuelsson, J.; Forssén, P.; Stefansson, M.; Fornstedt, T. Anal. Chem. 2004, 76, 953-958) that this peak consists of displaced plateau molecules; the injected molecules (mass peak) elute later, together with a deficiency of plateau molecules and are therefore not detected. In this article, we investigated what happens if a large rather than a small excess of solute molecules is injected. To study this systematically, the experimental method involved an enantiomer pair in an achiral separation system. It was found that the invisible mass peak was extremely deformed and that its shape depended on the amount of excess injected, the eluent concentration, and the column length. Depending on these operational conditions, the mass peak changed from a classical Langmuirian (tailing) to an anti-Langmuirian (leading) shape, with deformed shapes observable in the transition. The visible, overloaded perturbation peak was always Langmuirian, regardless of the mass peak shape.

Journal Article↗

PubMed2006

Discovery of invisible extra fronts in single-component frontal analysis in liquid chromatography.

Frontal analysis (FA) is often used in the "staircase mode", in which the solute concentration in the eluent increases stepwise. We demonstrate here in the single-component case, that all eluted breakthrough curves (fronts) for the second and subsequent steps consist solely of displaced plateau molecules. The newly introduced molecules (i.e., the introduced mass) instead elute later, in a breakthrough front hidden from detection, i.e., the mass front. These effects were studied using experimentally verified numerical calculations, the mass fronts being visualized using an enantiomer pair in an achiral separation system. Notably, the mass front displays no self-sharpening effects, even under strongly nonlinear conditions. Instead, the front is sigmoidal in shape.

Adsorption↗

PubMed2006

Analytical characterization of chiral drug-protein interactions: comparison between the optical biosensor (surface plasmon resonance) assay and the HPLC perturbation method.

Two modern, fundamentally different methods were used for a detailed investigation of enantioselective drug-protein interactions, a surface plasmon resonance (SPR)-based Biacore 2000 biosensor assay and the previously validated HPLC perturbation method (HPLC-PM). This is the first time SPR has been used for this purpose. The fundamental features of the two methods were investigated, and the consequences for operation and data evaluation were addressed. With HPLC-PM, chiral data could be obtained directly from the racemic mixture, whereas a separate analysis of each pure enantiomer was required to obtain chiral data with SPR. It was shown that if chirality is not attributed in the SPR analysis, misleading average racemic binding constants will be obtained. Both drug and protein consumption were considerably higher with HPLC-PM. HPLC-PM was found to be best suited for measurements of weak affinity interactions, whereas the SPR method was best for strong interactions. With both methods, the presence of DMSO in the samples severely affected the interactions, introducing errors. The binding of the beta-blockers alprenolol and propranolol to Cel7a cellulase was used as a model system. These methods gave results that agreed quite well qualitatively, but considerable quantitative deviations were sometimes obtained.

Adrenergic beta-Antagonists↗

PubMed2005

Accurate and rapid estimation of adsorption isotherms in liquid chromatography using the inverse method on plateaus.

The inverse method (IM) is an attractive approach for estimating adsorption isotherm parameters in liquid chromatography (LC), mainly due to its experimental simplicity and low sample consumption. This article presents a new experimental approach, the inverse method on plateaus (IMP), which uses elution profiles on concentration plateaus together with IM. This approach enabled us to obtain very accurate adsorption isotherms that agreed well with those estimated by means of frontal analysis over the entire concentration range under consideration. IMP is recommended when accurate adsorption isotherm estimates are required, and standard IM is insufficient.

Adsorption↗

PubMed2005

Investigation of the adsorption behaviour of a chiral model compound on a tartardiamide-based network-polymeric chiral stationary phase.

The adsorption behaviour of the enantiomers of 2-phenylbutyric acid on the chiral stationary phase (CSP) Kromasil CHI-TBB was studied using hexane/MTBE (90/10) as eluent. Adsorption isotherms were acquired at 40 different enantiomer concentrations in the interval between 7.6 microM and 305 mM, an approximately 40,000-fold dynamic range. The adsorption data fitted well to the bi-Langmuir model, indicating a heterogeneous surface with two different types of adsorption sites having different equilibrium constants and capacities; namely one chiral site and one non-chiral site. A comparison with earlier adsorption studies on modern CSPs revealed that the capacity value of the "true" chiral site of Kromasil CHI-TBB is the largest reported so far. The elution profiles simulated with these parameters show excellent agreement with the corresponding experimental profiles. Guidelines for comparisons of loading capacities of CSPs are presented.

Adsorption↗

PubMed2004

Validation of the accuracy of the perturbation peak method for determination of multicomponent adsorption isotherm parameters in LC.

The isotherm parameters were for the first time determined for a quaternary mixture. This was done by the perturbation peak (PP) method using racemic mixtures of methyl and ethyl mandelate enantiomers. One complication with the PP method is that the traditional blank injection technique makes all perturbation peaks, except one, vanish at moderately nonlinear concentration plateaus. Therefore, we devised a new injection technique that made all four peaks on a quaternary component concentration plateau detectable, thereby making the determination of multicomponent competitive isotherm parameters possible. The measured quaternary perturbation data fitted well to the bi-Langmuir isotherm model and excellent agreement was found between experimental and simulated single-component and multicomponent profiles, thus validating the method and the determined isotherm parameters. The method (i) is valuable for computer-assisted optimization of preparative chiral chromatography and (ii) opens the possibility of quantifying competitive drug-target interactions for chiral drugs directly on racemic mixtures, which today is impossible with any nonlabeled technique including surface plasmon resonance technology.

Adsorption↗

PubMed2004

Validation of the accuracy of the perturbation peak method for determination of single and binary adsorption isotherm parameters in LC.

An analytical validation of the precision and accuracy of the perturbation peak (PP) method for determination of single and competitive thermodynamic isotherm parameters was performed using frontal analysis as a reference. The isotherm parameters of 11alpha-hydroxyprogesterone were determined in an achiral system and the isotherm parameters of (+)-methyl L-mandelate and (-)-methyl D-mandelate were determined in a chiral system, both for the single components and for the competitive binary mixture. The experimental errors in the PP method using different injection techniques were investigated, and we devised a new injection technique for the determination of competitive isotherm parameters that considerably reduced the experimental errors and also made both perturbation peaks detectable. We showed that the PP method with the new injection technique can be used to determine isotherm parameters directly from a racemic mixture. These parameters agreed well with those determined using several enantiomer ratios. Elution-band profiles simulated using the isotherm parameters showed excellent agreement with experimental profiles.

Chromatography, Liquid↗

PubMed2004

Experimental proof of a chromatographic paradox: are the injected molecules in the peak?

Forty years ago Helfferich and Peterson published an article in Science regarding a "paradoxical" behavior in nonlinear chromatography (Helfferich, F.; Peterson, D. L. Science 1963, 142, 661-662). They theoretically predicted that when an excess of sample molecules is injected into a chromatographic column that is equilibrated with a constant stream of identical molecules, the observed peak will not contain the injected molecules. Instead the observed peak will only contain molecules from the stream whereas the injected molecules will exit the column in a slower moving, "invisible" peak. They considered it paradoxical that a single injection in a single-component system could cause the successive elution of two peaks (Helfferich, F. J. Chem. Educ. 1964, 41, 410-413). In this study, the paradox is experimentally proven for the first time. Two different strategies were employed: (i) a radiochemical approach and (ii) a method based on the use of two enantiomers in a nonchiral separation system. The experiments were compared with computer simulations.

Journal Article↗

PubMed2003

Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model.

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.

Biotechnology↗

PubMed2003

Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone.

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

Biotechnology↗

PubMed2003

Theoretical and experimental study of binary perturbation peaks with focus on peculiar retention behaviour and vanishing peaks in chiral liquid chromatography.

The perturbation peak theory was recently developed for acquiring binary isotherm data using the perturbation method (PM) and it was applied for some chiral systems. However, the binary plateaus of these systems were only weakly to moderately nonlinear. In this article the perturbation theory for LC, is developed for both retention times and peak areas and is verified by systematic experiments over the whole range of non-linearity. Attention is focused on non-linear effects that complicate the proper use of the PM method under moderately to strongly non-linear conditions. A serious complication was that the second perturbation peak vanished already at moderate plateau concentrations. A solution to this problem based on a firm theoretical basis and verified experimentally is presented. We also investigated a peculiar retention dependence on the binary plateau concentration, as the retentions of the two perturbation peaks of the binary plateau was compared with the single plateau peak of the more retained enantiomer.

Analysis of Variance↗