| 1. |
- Elhamili, Anisa, et al.
(författare)
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Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries
- 2011
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 32:6-7, s. 647-658
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4×105plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.
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| 2. |
- Enmark, Martin, 1984-, et al.
(författare)
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Analytical and preparative separation of phosphorothioated oligonucleotides : Columns and ion-pair reagents
- 2020
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 412:2, s. 299-309
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Tidskriftsartikel (refereegranskat)abstract
- Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 520mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.
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| 3. |
- Enmark, Martin, et al.
(författare)
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Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase
- 2011
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1218:38, s. 6688-6696
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Tidskriftsartikel (refereegranskat)abstract
- An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate–adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate–adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.
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| 4. |
- Enmark, Martin, 1984-, et al.
(författare)
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Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides
- 2019
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 411:15, s. 3383-3394
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Tidskriftsartikel (refereegranskat)abstract
- This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.
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| 5. |
- Enmark, Martin, 1984-, et al.
(författare)
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Investigation of robustness for supercritical fluid chromatography separation of peptides : Isocratic vs gradient mode
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1568, s. 177-187
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Tidskriftsartikel (refereegranskat)abstract
- We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO2-MeOH-H2O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system. To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer.
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| 6. |
- Fornstedt, Torgny, et al.
(författare)
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Deformations of overloaded bands under pH-stable conditions in reversed phase chromatography
- 2011
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1218:15, s. 1966-1973
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Tidskriftsartikel (refereegranskat)abstract
- It has recently been demonstrated, using mathematical models, how peculiar overloaded band profiles of basic compounds are due to the local pH in the column when using low capacity buffers. In this study, overloaded peak shapes resulting after injection of carefully pH matched samples close to the pKa of the chosen solute are investigated primarily on two columns; one hybrid silica C18 column (Kromasil Eternity) and one purely polymeric column (PLRP-S), the latter lacking C18 ligands. It was found that distorted peaks of the basic test compound appear even though there is no difference in pH between the injected sample solution and the eluent; the previous explanation to why these effects occur is based on a pH mismatch. Thus, the unusual band shapes are not due to an initial pH difference. Furthermore, it was observed that the effect does not appear on polymeric columns without C18 ligands, but only on columns with C18 ligands, independently of the base matrix (silica, hybrid silica, polymeric)
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| 7. |
- Fornstedt, Torgny, 1957-, et al.
(författare)
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Injection profiles in liquid chromatography. I. A fundamental investigation
- 2010
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Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier. - 0021-9673 .- 1873-3778. ; 1217:26, s. 4306-4312
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Tidskriftsartikel (refereegranskat)abstract
- This is a fundamental experimental and theoretical investigation on how the injection profile depends on important experimental parameters. The experiments revealed that the injection profile becomes more eroded with increased (i) flow rate, (ii) viscosity of the eluent, (iii) size of the solute, (iv) injection volume and (v) inner diameter of the injection loop capillary. These observations cannot be explained by a 1D-convection-diffusion equation, since it does not account for the effect of the parabolic flow and the radial diffusion on the elution profile. Therefore, the 1D model was expanded into a 2D-convection-diffusion equation with cylindrical coordinates, a model that showed a good agreement with the experimental injection profiles dependence on the experimental parameters. For a deeper understanding of the appearance of the injection profile the 2D model is excellent, but to account for injection profiles of various injection volumes and flow rates in preparative and process-chromatography using computer-optimizations, a more pragmatic approach must be developed. The result will give guidelines about how to reduce the extra-column variance caused by the injection profile. This is important both for preparative and analytical chromatography; in particular for modern analytical systems using short and narrow columns
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| 8. |
- Forssén, Patrik, 1966-, et al.
(författare)
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Injection profiles in liquid chromatography II : Predicting accurate injection-profiles for computer-assisted preparative optimizations
- 2011
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Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier. - 0021-9673 .- 1873-3778. ; 1218:34, s. 5794-5800
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Tidskriftsartikel (refereegranskat)abstract
- In computer assisted optimization of liquid chromatography it has been known for some years that it is important to use experimental injection profiles, instead of rectangular ones, in order to calculate accurate elution bands. However, the incorrectly assumed rectangular profiles are still mostly used especially in numerical optimizations. The reason is that the acquisition of injection profiles, for each injection volume and each flow rate considered in a computer-assisted optimization requires a too large number of experiments. In this article a new function is proposed, which enables highly accurate predictions of the injection profiles and thus more accurate computer optimizations, with a minimum experimental effort. To model the injection profiles for any injection volume at a constant flow rate, as few as two experimental injection profiles are required. If it is desirable to also take the effect of flow rate on the injection profiles into account, then just two additional experiments are required. The overlap between fitted and experimental injection profiles at different flow rates and different injection volumes were excellent, more than 90%, using experimental injection profiles from just four different injection volumes at two different flow rates. Moreover, it was demonstrated that the flow rate has a minor influence on the injection profiles and that the injection volume is the main parameter that needs to be accounted for.
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| 9. |
- Forssén, Patrik, 1966-, et al.
(författare)
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Optimization strategies accounting for the additive in preparative chiral liquid chromatography
- 2012
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1269:SI, s. 279-286
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Tidskriftsartikel (refereegranskat)abstract
- This study is an in-depth investigation on how numerical optimization strategies that also account for the additive type and concentration, in preparative batch chromatography, should be performed. As a model system, the separation of Z-(R,S)-2-aminobutyric acid enantiomers on a quinidine carbamate-based chiral stationary phase in polar organic mode was used, with different additive strengths of acetic acid or hexanoic acid in methanol. The inverse method was used to determine the competitive adsorption isotherm parameters for the enantiomers and the additives. Three different optimization strategies were examined: (1) injection volume optimization, (2) optimization of injection volume and additive concentration, and (3) full optimization including injection volume, additive concentration, sample concentration and flow rate. It was concluded that (i) it is important to incorporate the additive concentration in the optimization procedure to achieve the highest production rates, (ii) the full optimization strategy had the overall best results, and (iii) the selection of additive is very important (here acetic acid additive was superior to the hexanoic acid additive). By including the additive in the adsorption model and in the numerical optimization it is not only possible to achieve higher production rates but also to properly select the additive that is most advantageous for the specific separation problem.
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| 10. |
- Multia, Evgen, et al.
(författare)
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Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions
- 2017
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 518, s. 25-34
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Tidskriftsartikel (refereegranskat)abstract
- Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.
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