Simultaneous determination of ten nonsteroidal anti-inflammatory drugs from drinking water, surface water and wastewater using micro UHPLC-MS/MS with on-line SPE system.

A simple, accurate and sensitive micro UHPLC-MS/MS method was developed and validated for the simultaneous determination of 10 nonsteroidal anti-inflammatory drugs (NSAIDs) from different environmental matrices. The micro LC ‒ on-line SPE method described in this study allowed to determine the selected drugs at ultra-trace levels without the most commonly used complex off-line SPE sample preparation procedures. The presented method is capable of reaching satisfactory low LOQ values with analysing the sample directly after being diluted with water. In order to attain high sensitivity, mass spectrometry was carefully optimized for the analysis of the drugs. Fenoprofen, flurbiprofen and naproxen were found to produce CO2 loss during ionization, forming intense [M-H-CO2]- ions instead of [M-H]-. All the other compounds were analyzed through their [M+H]+ and [M-H]- ions. Effect of mobile phase pH on ionization was also studied. Lower pH resulted in higher ion intensities. For this reason, a reversed phase chromatographic separation was applied at pH 3.1 with formic acid at concentration of 0.01%. Matrix effects have been evaluated during validation and sample dilution was optimized focusing on the lowest achievable LOQ values. Analytes were determined from drinking water directly, from surface water and wastewater following dilution with purified water by 2 : 8 (v/v) and 1 : 9 (v/v), respectively. Finally, the method was applied to real sample analysis.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement.

Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass spectrometric peak tracking.

An older method for terazosin was reworked in order to reduce the analysis time from 90min (2×45min) to below 5min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified impurities separately. The reason of the different methods is that the retention of two impurities is not adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by different method than the other specified impurities. In our new method we could improve the retention with pH elevation using a new type of stationary phases available for high pH applications. Also a detection wavelength could be selected that is appropriate for the detection and quantification of all impurities. The method development is the bottleneck of liquid chromatography even today, when more and more fast chromatographic systems are used. Expert knowledge with intelligent programs is available to reduce the time of method development and offer extra information about the robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good alternative way to identify a certain peak in different chromatograms is the molecular mass of the compound, due to its high specificity. Liquid Chromatography-Mass Spectrometry (LC-MS) is now a routine technique and increasingly available in laboratories. In our experiment for the resolution- and retention modeling the DryLab4 method development software (Version 4.2) was used. In recent versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking technology. The modelled and measured chromatograms showed excellent correlations. The average retention time deviations were ca. 0.5s and there was no difference between the predicted and measured Rs,crit -values.

Separation of Isomers of JWH-122 on Porous Graphitic Carbon Stationary Phase with Non-Aqueous Mobile Phase Using Intelligent Software.

Cannabimimetic compounds have gained an increasing attention from the forensic community during the past few years. The present study was aimed to develop a liquid chromatographic separation method for the analysis of JWH-122 and its methyl isomers. In Hungary, JWH-122 is scheduled as a narcotic compound and its methyl isomers fall into the new psychoactive substance category, attracting significantly milder punishment than JWH-122 does. According to our best knowledge, gas chromatography or reversed phase liquid chromatography coupled with mass spectrometry could not be applied for separation and selective determination of methyl-naphthoyl indol isomers. In this study, we aimed to develop a high performance liquid chromatography method with UV and mass spectrometric detection for the separation of JWH-122 and all its possible isomers, depending on the position of methyl group on the naphthyl frame. Different reversed phase columns were used. Alkyl-modified silica with different selectivity and morphology with different mobile phase composition cannot be applied for separation of JWH-122 isomers. Porous graphitic carbon (PGC) column was used for separation of banned JWH-122 and each of its methyl isomers. In method development, a Quality by Design approach is presented for modeling the retention of the compounds. According to our knowledge, this is the first time reporting the use of intelligent software to estimate the retention on PGC material and using non-aqueous conditions. Retention times predicted by two program packages (STATISTICA® and DryLab®) are compared. The possibilities and limitations of the software modeling in the conditions described above are also evaluated.

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions.

Challenges in liquid chromatographic characterization of proteins.

Various liquid chromatographic techniques are considered standard analytical methods in proteins characterization. These methods provide essential information for drug approval, for biological and life sciences. On the other hand, there are some issues and challenges which have to be taken into account when analyzing these biopharmaceuticals. The aim of this review to summarize the most recent knowledge relating to the following topics: i) sample stability and complexity ii) adsorption problems: instrument inertness iii) adsorption problems: recovery from the stationary phase and iv) challenges in method development. This information is supposed to help practicing chromatographers in the emerging field of therapeutic protein chromatography.

Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column.

The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation ("inverse gradient elution" on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, i.d.; 5 µm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r = 0.9987) over the concentration range from 1 to 1000 nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0-10.2% and -6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1 nM (0.446 ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides.

Establishing column batch repeatability according to Quality by Design (QbD) principles using modeling software.

Column technology needs further improvement even today. To get information of batch-to-batch repeatability, intelligent modeling software was applied. Twelve columns from the same production process, but from different batches were compared in this work. In this paper, the retention parameters of these columns with real life sample solutes were studied. The following parameters were selected for measurements: gradient time, temperature and pH. Based on calculated results, batch-to-batch repeatability of BEH columns was evaluated. Two parallel measurements on two columns from the same batch were performed to obtain information about the quality of packing. Calculating the average of individual working points at the highest critical resolution (R(s,crit)) it was found that the robustness, calculated with a newly released robustness module, had a success rate >98% among the predicted 3(6) = 729 experiments for all 12 columns. With the help of retention modeling all substances could be separated independently from the batch and/or packing, using the same conditions, having high robustness of the experiments.

Systematic evaluation of mobile phase additives for the LC-MS characterization of therapeutic proteins.

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive for the analysis of proteins in reversed phase liquid chromatography (RPLC). Due to its interesting features, it provides symmetrical and narrow peak shapes for proteins, but decreases mass spectrometric sensitivity through ion-pairing and spray destabilizing. Since RPLC-MS is an important technique for the characterization of proteins, some alternative MS-compatible mobile phases may be required. The aim of this study was to evaluate various acidic and basic mobile phase additives for the LC-MS analysis of therapeutic proteins possessing molecular weight between 5 and 150kDa. At the end, 10mM formate buffer pH 3 was found to be the most promising alternative, since it provided acceptable peak shapes in most cases, together with an average improvement of MS sensitivity by 5-times, compared to TFA.

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach.

The cation exhange pH gradient approach was evaluated for the characterization of 10 model monoclonal antibodies including panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, adalimumab, denosumab and ofatumumab. This work shows that retention and resolution can be modelled in cation exchange pH gradient mode, based on only four initial runs (i.e. two gradient times and two mobile phase temperature). Only 6h were required for a complete method optimization when using a 100 mm × 4.6 mm strong cation exchange column. The accuracy of the predictions was excellent, with an average difference between predicted and experimental retention times of about 1%. The 10 model antibodies were successfully eluted in both pH and salt gradient modes, proving that both modes of elution can be considered as multi-product charge sensitive separation methods. For most of the compounds, the variants were better resolved in the salt gradient mode and the peak capacities were also higher in the salt gradient approach. These observations confirm that pH gradient approach may be of lower interest than salt gradient cation exchange chromatography for antibody characterization.

Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach.

Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.

Recovery of Proteins Affected by Mobile Phase Trifluoroacetic Acid Concentration in Reversed-Phase Chromatography.

It was found that recoveries of proteins depend on trifluoroacetic acid concentration in the mobile phase and showed maximum in the range of 0.01-0.1 v/v%. Transferrin and lysozyme were used to evaluate the recoveries of proteins from dedicated reversed-phase columns. Different types of reversed-phase columns were evaluated, such as core shell type materials (Aeris Widepore with C4, C8 and C18 modification) as well as fully porous hybrid particles (Waters BEH, modified with C4 and C18 alkyl chains). Recoveries ranged between 60.7-95.2% for transferrin and 72.1-99.8% for lysozyme. Based on the data presented, at least two different adsorption effects, the well-known hydrophobic and silanophilic/polar interaction might influence the recovery. In addition to this, conformational effects due to ion pairing with the acidic mobile phase additive might change them.

Estimation of the effects of longitudinal temperature gradients caused by frictional heating on the solute retention using fully porous and superficially porous sub-2µm materials.

In this study, the retention changes induced by frictional heating were evaluated for model small compounds (150-190Da) and a small protein, namely insulin (5.7kDa). For this purpose, the effect of longitudinal temperature gradient caused by frictional heating was experimentally dissociated from the combined effect of pressure and frictional heating, by working either in constant and variable inlet pressure modes. Various columns packed with core-shell and fully porous sub-2µm particles were tested. It appears that frictional heating was less pronounced on the column packed with smallest core-shell particles (1.3µm), compared to the ones packed with core-shell and fully porous particles of 1.7-1.8µm. This observation was attributed to the low permeability of this material and the fact that it can only be employed in a restricted flow rate range, thus limiting the generated heat power. In addition, the thermal conductivity of the solid silica core of superficially porous particles (1.4W/m/K) is known to be much larger than that of fully porous silica. Then, the heat dissipation is improved. However, if systems with higher pressure capability would be available and the mechanical stability of 1.3µm core-shell material was extended to e.g. 2000bar, the retention would be more severely impacted. At 2000bar, ∼4.4W heat power and +30°C increase at column outlet temperature is expected. Last but not least, when analyzing large molecules, the impact of pressure overcomes the frictional heating effects. This was demonstrated in this study with insulin (∼5.7kDa).

Reliability of computer-assisted method transfer between several column dimensions packed with 1.3-5µm core-shell particles and between various instruments.

In this contribution, the possibility to automatically transfer RPLC methods between different column dimensions and instruments was evaluated using commercial modelling software. The method transfer reliability was tested with loratadine and its 7 related pharmacopeial impurities. In this study, state-of-the-art columns packed with superficially porous particles of 5, 2.6, 1.7 and 1.3µm particles were exclusively employed. A fast baseline separation of loratadine and related impurities (Rs,min=2.49) was achieved under the best analytical conditions (i.e. column of 50mm×2.1mm, 1.3µm, 10-90% ACN in 5min, T=40°C, pH=3, F=0.5ml/min). This optimal method was successfully tested on columns packed with other particle sizes, namely 1.7 and 2.6µm, to reduce pressure drop. The selectivities and retentions remained identical, while the peak widths were logically wider, leading to a reduction of peak capacity from 203 to 181 and 159 on the 1.3, 1.7 and 2.6µm particles, respectively. On the minimum, the resolution was equal to 1.54 on the 50mm×2.1mm, 2.6µm stationary phase. Next to this, the method was transferred to columns of different lengths, inner diameters and particle sizes (100mm×3mm, 2.6µm or 150mm×4.6mm, 5µm). These columns were used on other LC instruments possessing larger dwell volumes. The modelling software employed for developing the original method was able to calculate the new gradient conditions to be used. The accuracy of prediction was excellent, as the average retention time errors between predicted and observed chromatograms were -0.11% and 0.45% when transferring the method to 100mm×3mm and 150mm×4.6mm columns, respectively. This work proves the usefulness and validity of HPLC modelling software for transferring methods between different instruments, column dimensions and/or flow rates.

Reliability of simulated robustness testing in fast liquid chromatography, using state-of-the-art column technology, instrumentation and modelling software.

The goal of this study was to evaluate the accuracy of simulated robustness testing using commercial modelling software (DryLab) and state-of-the-art stationary phases. For this purpose, a mixture of amlodipine and its seven related impurities was analyzed on short narrow bore columns (50×2.1mm, packed with sub-2µm particles) providing short analysis times. The performance of commercial modelling software for robustness testing was systematically compared to experimental measurements and DoE based predictions. We have demonstrated that the reliability of predictions was good, since the predicted retention times and resolutions were in good agreement with the experimental ones at the edges of the design space. In average, the retention time relative errors were <1.0%, while the predicted critical resolution errors were comprised between 6.9 and 17.2%. Because the simulated robustness testing requires significantly less experimental work than the DoE based predictions, we think that robustness could now be investigated in the early stage of method development. Moreover, the column interchangeability, which is also an important part of robustness testing, was investigated considering five different C8 and C18 columns packed with sub-2µm particles. Again, thanks to modelling software, we proved that the separation was feasible on all columns within the same analysis time (less than 4min), by proper adjustments of variables.

Determination of tetracyclines in pig and other meat samples using liquid chromatography coupled with diode array and tandem mass spectrometric detectors.

Two high performance liquid chromatographic methods (HPLC-DAD and LC-MS/MS) were developed to analyze tetracycline (TC) residues in pig meat (pork) samples. The method involved a sample preparation using a solid-liquid extraction (SLE) by McIlvaine buffer, followed by a solid-phase extraction (SPE) clean-up using Strata-XL cartridges. The developed sample clean-up resulted in a selective chromatogram in the HPLC-DAD separation and a reduced matrix effect (ME) in LC-MS/MS analysis. Moreover, HPLC columns packed with core-shell particles were tested for separation, which further enhanced the sensitivity and the selectivity of determinations. The validation of the methods for pig samples was carried out according to European Union 2002/657/EC decision. In addition, validation was also performed for bovine, chicken, and turkey meat samples using HPLC-DAD method. The performance characteristics of determinations were evaluated with both spiked and incurred samples, and were systematically compared. LC-MS/MS technique was found to be more accurate for spiked samples; however, HPLC-DAD method resulted in more reliable concentrations for incurred samples.

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.

Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.

Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography-tandem mass spectrometry.

This paper describes a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the analysis of three stanozolol metabolites (16ß-hydroxystanozolol, 3'-hydroxystanozolol, and 4ß-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC-MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core-shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15µg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.

Possibility of large volume injection and band focusing in UHPLC.

New shell-type stationary phases are widely used in fast chromatographic measurements. These columns provide more efficient separation, when applied in a conventional high-performance liquid chromatography instrument, than columns with fully porous particles, and the volume overload of core-shell particles is 60% of the value obtained for fully porous particles. Additionally, to achieve adequate sensitivity, the injection volume cannot be significantly decreased. This study presents a systematic evaluation of the possibilities of large volume injection onto columns packed with 2.6 µm Kinetex C18 shell particles. The effect of volume overload on performance of columns with different lengths (50, 100 and 150 mm) is studied. Column efficiency is compared under isocratic, pulse gradient and gradient conditions. The application of large volume injection in practice is also reported. The most suitable among the tested large volume injection techniques was the gradient elution, which was applied to determine amino acid enantiomers from fruit juice.

Simultaneous determination of eight corticosteroids in bovine tissues using liquid chromatography-tandem mass spectrometry.

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 500 V 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 ß-epimer, betamethasone. Dexamethasone and betamethasone were eluted within 12 min 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. 1 µg/kg, respectively.