[Show abstract][Hide abstract] ABSTRACT: Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ∼250 Å and macropores of ∼ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ∼300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.
Analytical and Bioanalytical Chemistry 01/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recombinant monoclonal antibodies (mAbs) have become particularly relevant for the treatment of autoimmune diseases or cancers. Because of their inherent complexity and for safety reasons, there is a need to develop powerful analytical methods to provide detailed characterizations of mAbs.
The aim of the present review is to detail the state-of-the-art of analytical strategies for mAb characterization. It focuses on the most important separation techniques used in this field, specifically, the chromatographic and electrophoretic approaches and their combination with mass spectrometry (MS). Thanks to recent improvements in separation science and MS devices, mAbs can be analyzed more easily. However, there is still a need to find new approaches that avoid adsorption issues.
[Show abstract][Hide abstract] ABSTRACT: This paper presents new reversed phase liquid chromatographic methods (HPLC-FLD and LC-MS/MS) for the quantification of sulfonamides in spiked and incurred honey samples. The sample preparation was optimized using Oasis HLB (hydrophilic–lipophilic balance) solid-phase extraction (SPE) cartridge. Elutions of sulfonamides were carried out under acidic, neutral, and basic conditions using methanol. Recoveries under acid condition were in the range from 66.8–90%, which were approximately 10% higher than those obtained under other conditions. The sample clean-up was also tested using Strata-XL cartridges. The HPLC-FLD separation was performed using a Varian C18 column and a ternary (methanol-acetonitrile-phosphate buffer, pH 5) mobile phase resulting good selectivity for the determination. The robustness of the ternary gradient method was evaluated by computer simulation (DryLab). LC-MS/MS separation was carried out on a Kinetex XB core-shell type HPLC column that enabled a low limit of detection (0.01–0.5 µg/kg) and faster separation (6 min). The developed methods were validated in accordance with the European Union Commission Decision 2002/657/EC and were applied successfully for more than four hundred honey samples (under a national monitoring program). The concentrations of sulfadimethoxine, sulfachloropyridazine, and trimethoprim residues in samples were found in a concentration range from 0.03 up to 686 µg/kg.
Journal of Liquid Chromatography & Related Technologies 01/2013; 36(8). · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A possible complication of ultra-high pressure liquid chromatography (UHPLC) is related to the effect of pressure and mobile phase velocity on the retention properties of the analytes. In the present work, numerous model compounds have been selected including small molecules, peptides, and proteins (such as monoclonal antibodies). Two instrumental setups were considered to attain elevated pressure drops, firstly the use of a post-column restrictor capillary at low mobile phase flow rate (pure effect of pressure) and secondly the increase of mobile phase flow rate without restrictor (i.e. a combined effect of pressure and frictional heating). In both conditions, the goal was to assess differences in retention behaviour, depending on the type or character of the analyte. An important conclusion is that the effect of pressure and mobile phase velocity on retention varied in proportion with the size of the molecule and in some cases showed very different behaviour. In isocratic mode, the pure effect of pressure (experiments with a post-column restrictor capillary) induces an increase in retention by 25-100% on small molecules (MW<300g/mol), 150% for peptides (∼1.3kDa), 800% for insulin (∼6kDa) and up to >3000% for myoglobin (∼17kDa) for an increase in pressure from 100bar up to 1100bar. The important effect observed for the isocratic elution of proteins is probably related to conformational changes of the protein in addition to the effect of molecular size. Working in gradient elution mode, the pressure related effects on retention were found to be less pronounced but still present (an increase of apparent retention factor between 0.2 and 2.5 was observed).
Journal of Chromatography A 11/2012; · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes a newly developed method for the simultaneous determination of eight corticosteroid residues in bovine muscle, liver and kidney samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The determination of methylprednisone, the main metabolite of methylprednisolone, in bovine tissues using LC-MS/MS is carried out for the first time. The method development demonstrates that the pH is important in optimizing the sample preparation. Tests performed using different solid-phase extraction (SPE) cartridges were enabled to produce conditions for reducing the matrix effects (ion suppression and enhancement) of analysis. Acidic condition and mixed-mode cation exchange SPE columns resulted in the most suitable clean-up for muscle and liver, and also yielded acceptable results for kidney. The enhanced sample clean-up resulted in excellent clear baselines of ion transitions, and therefore, a higher delta electron multiplier voltage (ΔEMV) could be set in the MS/MS detector. The application of 500V of ΔEMV improved the signal responses, however, the noise level did not change, and consequently, the overall sensitivity and analytical limits (limit of detection, limit of quantification) could be enhanced. In the HPLC separation, the recently introduced Kinetex phenyl-hexyl core-shell type column was used that enabled baseline separation for dexamethasone and its β-epimer, betamethasone. Dexamethasone and betamethasone were eluted within 12min and such reduced retention, obtained with core-shell HPLC type column, further enhanced the sensitivity. The method was validated according to the European Union (EU) 2002/657/EC Decision; the studied parameters met the EU standards. The decision limits and limit of detections were calculated in each matrix for all corticosteroids and varied from 0.01 to 13.3μg/kg and from 0.01 to 0.1μg/kg, respectively.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2012; 906:75-84. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present contribution, columns packed with fully porous widepore 1.7μm particles (Acquity BEH300) and widepore core-shell 3.6μm particles (Aeris WP) were evaluated for the separation of model and therapeutic proteins of varying sizes, hydrophobicity and isoelectric points. Two types of bonding were compared, namely C4 and C18 in a systematic way. The kinetic performance of these stationary phases was evaluated in a previous paper hence this new work focuses on their retention behaviour, loading capacity and selectivity. Using the Tanaka tests, model proteins, and other confirmatory experiments, it is highly probable that with proteins, strong interaction mechanisms were predominant on the Aeris WP while the hydrophobic interaction was the driving force of the retention on the Acquity BEH300 material. This explained why, despite the lower pore volume of the Aeris WP material, the apparent retention factors of proteins possessing both hydrophobic and charged amino acids residues were very close on the four investigated columns. In terms of peak widths, values for proteins were similar for all the tested stationary phases, despite the probable strong ion exchange mechanisms of Aeris WP column. This could be explained by the excellent mass transfer characteristics afforded by the thin porous layer (∼0.2μm) at the surface of the particle which probably compensates for the slow secondary ionic interaction kinetics. The loading capacity was also evaluated on all the four widepore columns, using model proteins. On average, approximately 2-4 times higher amount of proteins can be injected on the fully porous BEH300 compared to the core-shell Aeris WP columns when avoiding 10% change in peak width or in tailing. However, this result could be strongly influenced by the nature and shape of the protein, its hydrophobicity, folding, size and number of charges. Finally, all of these columns were employed for the highly efficient separation of a therapeutic protein (interferon-α-2A) and some closely related proteins and showed excellent performance and selectivity. This result confirms that RPLC gained interest in the biopharmaceutical field as it provides significantly better peak widths than size-exclusion or ion-exchange and inherent compatibility with MS.
Journal of Chromatography A 06/2012; 1252:90-103. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) are an emerging class of therapeutic agents that have recently gained importance. To attain acceptable kinetic performance with mAbs in reversed phase liquid chromatography, there is a need to work with the last generation of wide-pore sub-2μm fully porous or core-shell particles stationary phases. In addition, temperature in the range 60-90°C was found to be mandatory to limit adsorption phenomenon of mAbs and their fragments. A generic method development strategy was proposed to account for the selectivity, efficiency, recovery, and the possible thermal degradation. This study also demonstrated that the gradient steepness and temperature cannot be optimized using van't Hoff type linear models. Similarly, the common linear solvent strength model also generated some error in predicting the retention times. In contrast, when quadratic models were employed, the prediction accuracy of retention times was found to be excellent (relative error between 0.5 and 1%) using a reasonable number of experiments (9 or 6 experiments for optimization of gradient time and temperature, which requires between 6 and 8h). Two separations of mAbs fragments were performed to demonstrate the reliability of the quadratic approach.
Journal of pharmaceutical and biomedical analysis 06/2012; 70:158-68. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent reversed-phase wide-pore stationary phases were evaluated for the separation of intact monoclonal antibodies and their fragments. Two types of stationary phases were tested: Phenomenex Aeris Widepore, with 3.6 μm core-shell particles and Waters Acquity BEH300 with 1.7 μm fully porous particles. A systematic investigation was carried out using model IgG1 and IgG2 antibodies, namely rituximab, panitumumab, and bevacizumab. It appeared that adsorption of these antibodies on the stationary phase was significantly higher compared to proteins of equivalent size. The adsorption was particularly important for the intact antibodies of 150 kDa and for the largest fragments of 50 to 100 kDa (i.e., heavy chain, -fraction of antigene-binding). The present study demonstrated an obvious relationship between adsorption phenomenon and the unwanted strong secondary interactions (ionic and hydrogen bond) of the stationary phase. Thus, adsorption was more pronounced on the Aeris column because of the stronger ion exchange contribution of this stationary phase. Using C4 phase instead of C18 at 50-70°C, there is a slight reduction (5-20%) in adsorption. Two solutions were proposed to decrease the strength of secondary interactions and thus resolve (or at least diminish) adsorption issue. First, increasing mobile phase temperature up to 80-90°C appeared as a promising solution. However, temperature should be used with caution as it can partially damage large biomolecules. A compromise between residence time and temperature should be found. Second, it is recommended to add a small amount of an ancillary solvent, such as n-butanol to the mobile phase. Indeed, the hydroxyl group of n-butanol probably interacts with water adsorbed on the residual silanol groups "to shield" silanols.
Journal of Separation Science 06/2012; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.
Journal of pharmaceutical and biomedical analysis 03/2012; 69:9-27. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2 μm particles or shell particles. In this context, a new wide-pore core-shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2 μm solid inner core surrounded by a 0.2 μm porous layer (total particle size of 3.6 μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h(min) values of the Aeris WIDEPORE column were determined for small model compounds. The h(min) values were equal to 1.7-1.8 and 1.4 for the 2.1 and 4.6 mm I.D. columns, respectively, which are in agreement with the values reported for other core-shell materials. In the case of a small protein Insulin (5.7 kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7-66.8 kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8 kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t(grad)<6 min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.
Journal of Chromatography A 03/2012; 1236:177-88. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Columns packed with sub-2 μm totally porous and sub-3 μm core-shell particles are very widespread nowadays to conduct fast and efficient separations. In order to carry out really fast separations, short (5 cm long) columns are very popular today. The goal of this paper is to review the recent possibilities in fast or "ultra-fast" HPLC by applying short and narrow bore columns packed with modern core-shell and very fine fully porous particles. Efficiency data obtained with these recently commercialized columns from the past few years are collected, discussed and compared in terms of potential separation time and efficiency. The reasons of the success of these columns are presented. This paper also shows that theoretically expected efficiency is sometimes compromised in practical work especially in the case of narrow bore columns. The extra-column dispersion of a given LC system can also dramatically decrease the performance of small columns. It is not possible to utilize the real efficiency of these small columns in spite of their really high intrinsic separation power.
Journal of Chromatography A 03/2012; 1228:57-71. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A fast liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is developed to determine lincomycin (LM) in honey, muscle, milk, and egg. Samples are cleaned-up at pH 4.7 using Strata-X-C mixed-mode polymeric strong cation exchange solid-phase extraction (SPE) cartridges, which could selectively adsorb the lincomycin from matrices under the acidic condition. LM is separated on the recently introduced Kinetex XB core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water/acetonitrile (93/7, v/v, pH 2.6) at a flow rate of 0.7 mL/min. The subsequent MS/MS detection has decreased ion effect, which allows the limit of detection (LOD) of LM for honey to be 0.05 µg/kg for honey and 0.5 µg/kg for muscle, milk, and egg. These LODs are much lower than those reported previously. The other main advantage of the developed method is the analysis time of only 3.5 min, which is about three times shorter than other reported LC-MS-MS methods. Recoveries varies between 94.2% and 125.2% and in-house reproducibility ranges from 3.7% to 28.7%. The developed method is validated according to European Union (EU) Commission Decision 2002/657/EC using a matrix-comprehensive validation strategy. All studied analytical parameters fulfills the EU guidelines.
Journal of chromatographic science 03/2012; 50(3):190-8. · 0.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to identify and to quantify nitroimidazoles, metronidazole (MNZ), ronidazole (RNZ) and dimetridazole (DMZ) and their corresponding hydroxy metabolites, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMNNI) in plasma, milk, muscle, egg, honey and feed samples. The same sample clean-up procedure including a novel solid-phase extraction (SPE) on polymeric Strata-SDB cartridges was used for each matrix. The analytes were separated on Kinetex XB C-18 core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water/methanol (88/12, v/v, pH 2.6) at a flow rate of 0.7 ml/min. The main advantage of the developed method is that the analysis time of only 3 min, which is about three to ten times shorter than in other reported HPLC methods. The developed method was validated using a matrix-comprehensive in-house validation strategy. The matrix effect of LC-MS/MS analysis was also investigated. Results are presented from the successful application of the developed method to an incurred pork meat certified reference material and to incur porcine plasmas in a proficiency test in year 2011.
Journal of pharmaceutical and biomedical analysis 02/2012; 64-65:40-8. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Small columns packed with core-shell and sub-2 μm totally porous particles and monolith columns are very popular to conduct fast and efficient chromatographic separations. In order to carry out fast separations, short (2-5 cm) and narrow-bore (2-2.1 mm) columns are used to decrease the analyte retention volume. Beside the column efficiency, another significant issue is the extra-column band-spreading. The extra-column dispersion of a given LC system can dramatically decrease the performance of a small very efficient column. The aim of this study was to compare the extra-column peak variance contribution of several commercially available LC systems. The efficiency loss of three different type 5 cm long narrow bore, very efficient columns (monolith, sub-2 μm fully porous and sub-2 μm core-shell packing) as a function of extra-column peak variance, and as a function of flow rate and also kinetic plots (analysis time versus apparent column efficiency) are presented.
Journal of Chromatography A 08/2011; 1218(31):5286-91. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d(p)=2.7 μm) and structured (ρ=0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation.
[Show abstract][Hide abstract] ABSTRACT: A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core-shell column (100 mm × 3 mm, 2.6 μm) and methanol-water-trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10-60 μg/ml and unbound PEG in the range of 2-40 μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.
Journal of Chromatography A 10/2010; 1217(40):6258-66. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: At present sub-2 μm packed columns are very popular to accomplish rapid and efficient separations. Applying particles with shortened diffusion path to improve the efficiency of separation performs higher efficiency than it is possible with the totally porous particles having the same size. The advantages of sub-2 μm particles and shell particles are combined in the new Kinetex 1.7 μm particles. In this study a systematical evaluation of the efficiency and achievable analysis time obtained with 5 cm long narrow bore column packed with sub-2 μm core-shell particles (1.25 μm core diameter and 0.23 μm porous silica layer), and other type very efficient columns is presented. The efficiency of separation was investigated also for small pharmaceutical and large molecules (proteins). Van Deemter, Knox and kinetic plots are calculated. The results obtained with low molecular weight polar neutral analytes (272 g/mol, 875 g/mol), with a polypeptide (4.1 kDa) and with different sized proteins (18.8 kDa, 38.9 kDa and 66.3 kDa) are presented in this study. Moreover, particle size distribution, and average pore size (low-temperature nitrogen adsorption, LTNA) of the new very fine core-shell particles were investigated. According to this study, increased flow rates can be applied on sub-2 μm core-shell columns to accomplish very fast separations without significant loss in efficiency. The new sub-2 μm shell particles offer very high efficiency both for small and large molecule separation.
Journal of pharmaceutical and biomedical analysis 09/2010; 54(3):482-90. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Today sub-2 microm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 microm shell particles (1.9 microm nonporous core surrounded by a 0.35 microm porous shell, Kinetex, Core-Shell), packed with other shell-type particles (Ascentis Express, Fused-Core), totally porous sub-2 microm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW=270-430) and a high molecular weight one (MW approximately 900) was conducted. This study proves that the Kinetex column packed with 2.6 microm shell particles is worthy of rivaling to sub-2 microm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.
Journal of Chromatography A 06/2010; 1217(23):3642-53. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A fast and sensitive method has been developed for the specific determination of Polysorbate 80 (Tween 80) in liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and charged aerosol detection (CAD). The method was validated using a Poroshell 300SB-C18 column packed with 5 microm shell particles (75 mm x 2.1 mm) and acetonitrile-methanol-water-trifluoroacetic acid mobile phase at a flow rate of 0.65 ml/min. The rapid LC-CAD method is suitable for quantifying Polysorbate 80 in the range of 10-60 microg/ml in protein solutions within good manufacturing practices (GMPs) of the pharmaceutical industry.
Journal of pharmaceutical and biomedical analysis 03/2010; 52(5):672-9. · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cmx2.1mm columns packed with sub-2microm particles and computer simulation (DryLab((R)) package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3-5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.
Journal of Chromatography A 09/2009; 1216(45):7816-23. · 4.61 Impact Factor