Monolithic Silica Rods Research Articles

Fast Screening of Diol Impurities in Methoxy Poly(Ethylene Glycol)s (mPEG)s by Liquid Chromatography on Monolithic Silica Rods.

The determination of diol impurities in methoxy poly(ethylene glycol)s (mPEG)s is of high importance, e.g., in the area of pharmaceutical applications, since mPEGs are considered the gold standard—based on properties of biocompatibility, stealth effect against the immune system, and well-established procedures used in PEGylation reactions. Herein, we communicate a straightforward and fast approach for the resolution of the PEGdiol impurities in mPEG products by liquid chromatography on reversed-phase monolithic silica-rods. Thus, we utilize fine, in-house prepared and narrow dispersity mPEGs (Ð ≤ 1.1) and commercial PEGdiol standards as a reference. Most efficient analysis of diol impurities becomes possible with reversed-phase liquid chromatography that results in selective elution of the PEGdiol from mPEG macromolecule populations in partition/adsorption mode. We do this by a minimum selectivity of the population of macromolecules characterizing the narrow molar mass distributions of mPEG. Control experiments with intentionally added water at the start of the well-controlled mPEG synthesis via the living anionic ring opening polymerization of ethylene oxide clearly reconciled the existence of PEGdiol impurity in chromatographed samples. The here-demonstrated methodology allows for the resolution of diol impurities of less than one percent in elution times of only a few minutes, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the collected elution fractions. The unique combination of the open flow-through pore structure of the monolithic silica rods and resultant varying accessibility of C18-derivatized pore surfaces indicates beneficial properties for robust and end-group-specific adsorption/partition liquid chromatography of synthetic macromolecules.

Effect of polyethylene glycol on pore structure and separation efficiency of silica-based monolithic capillary columns

Monolithic silica materials (first unclad monolith rods, then monolithic capillary columns) were prepared using various amounts of polyethylene glycols (PEGs) with different molecular weight (MW). The monolith rods were used to examine the mesoporosity by argon physisorption technique, and the macroporosity by mercury intrusion porosimetry. Subsequently, silica-based monolithic capillary columns with an inner diameter of 100μm were produced using the same preparation conditions as used for the rods. The results obtained with the monolith rods showed the following important findings: (1) it is feasible to fabricate monolithic silica rods possessing macropore size of 0.5–1.4μm by tuning the amount of PEGs (independently of the MW), whereas the macropore volume and the mesoporosity remain similar. (2) the smallest macropore size (0.5μm) rod prepared with PEG having a MW=20,000g/mol provided a narrower macropore size distribution than with PEG with MW=10,000g/mol. The monolithic capillary columns produced with the different PEG type showed similar retention factors for hexylbenzene (k=2.3–2.4) and similar t0-based column permeability (Kv0=2.3–2.4×10−14m2) in 20:80% (v/v) water:methanol, as expected from the results obtained with the monolith rods. The column prepared with PEG of MW=20,000g/mol gave a plate height of H=4.0μm for hexylbenzene at an optimal linear velocity of u0=2.6mm/s in 20:80% (v/v) water containing 0.1% formic acid:acetonitrile. To the best of our knowledge, this is the lowest plate height ever recorded for a monolithic column. Comparing the kinetic performance at 30MPa shows that the best monolithic silica column obtained in the present study performs better than the second-generation monolithic silica columns developed up till now in the practically most relevant range of plate numbers (N≤40,000). In this range, the performance is now similar to that of 2.7μm core-shell particle columns.

Thermoresponsive hydrophobic copolymer brushes modified porous monolithic silica for high-resolution bioseparation

Thermoresponsive-hydrophobic copolymer brushes were prepared on porous monolithic silica rods through surface initiated ATRP. The monolithic silica can separate biomolecules with high resolution and in short analysis times.

Monolithic Silica Rods Grafted with Thermoresponsive Anionic Polymer Brushes for High-Speed Separation of Basic Biomolecules and Peptides

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Thermally Modulated Cationic Copolymer Brush on Monolithic Silica Rods for High-Speed Separation of Acidic Biomolecules

Poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethylmethacrylate(DMAEMA)-co-tert-butylacrylamide (tBAAm)), a thermoresponsive-cationic-copolymer, brush-grafted monolithic-silica column was prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive anion-exchange chromatography matrices. ATRP-initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. IPAAm, DMAEMA, and tBAAm monomers and CuCl/CuCl₂/Me₆TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator modified columns at 25 °C for 16 h. The constructed copolymer-brush structure on monolithic silica-rod surface was confirmed by X-ray photoelectron spectroscopy (XPS), elemental analysis, scanning electron microscopy (SEM) observation, and gel permeation chromatography (GPC) measurement of grafted copolymer. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. The cationic copolymer brush modified monolithic silica-rod columns were able to separate adenosine nucleotides with a shorter analysis time (4 min) than thermoresponsive copolymer brush-modified silica-bead-packed columns, because of the reduced diffusion path length of monolithic supporting materials. These results indicated that thermoresponsive cationic copolymer brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing acidic-bioactive compounds with a remarkably short analysis time.

Immobilization of invertase on silica monoliths with hierarchical pore structure to obtain continuous flow enzymatic microreactors of high performance

Monolithic silica rods of 4mm diameter and 2–5cm length, exhibiting very open and uniform 3D hierarchical pore structure of 35μm flow-through macropores and ca. 20nm mesopores were synthesized using the sol–gel processing combined with pore templating and phase separation. These monoliths were successfully converted into miniaturized multichannel continuous-flow reactors which (for a single rod) could operate at flow rates up to about 20mL/min at backpressure not exceeding 2.5bar. After covalent attachment of invertase, considered as a model enzyme, their potentials were tested in reaction of sucrose hydrolysis and the results were compared, using Michaelis–Menten kinetics, with the performance of mesoporous cellular foam (MCF)-bound invertase immobilized in the same way. The hydrolysis of sucrose appeared to proceed with maximum velocity over 1000 times faster in the monolithic bioreactor than in MCF-based slurry system, and invertase embedded in the mesopores of silica monoliths showed notably larger affinity (lower KM) to substrate than the native enzyme. The reactors stored at 4°C retained initial activity for at least 6week and no change in the microreactor performance was seen for at least 2week of continuous operation.

The current revolution in column technology: How it began, where is it going?

AbstractThis work revisits the exceptionally rapid evolution of the technology of chromatographic columns and the important progress in speed of analysis and resolution power that was achieved over the last ten years. Whereas columns packed with 10 and 5μm fully porous particles dominated the field for nearly thirty years (1975–2000), it took barely six years to see the commercialization of monolithic silica rods (2000), their raise to fame and decay to oblivion, the development of finer fully porous particles with size down to 1.7μm (2006), and of sub-3μm superficially porous particles (2006). Analysis times and plate heights delivered by columns packed with these recent packing materials have then been improved by more than one order of magnitude in this short period of time. This progress has rendered practically obsolete the age-old design of LC instruments. For low molecular weight compounds, analysts can now achieve peak capacities of 40 peaks in about 15s with a hold-up time of the order of 1.5s, in gradient elution, by operating columns packed with sub-3μm shell particles at elevated temperatures, provided that they use optimized high pressure liquid chromatographs. This is the ultimate limit allowed by modern instruments, which have an extra-column band broadening contribution of 7μL2 at 4.0mL/min and data acquisition rate of 160Hz. The best 2.1mm×50mm narrow-bore columns packed with 1.7μm silica core–shell particles provide peaks that have a variance of 2.1μL2 for k=1. Finally, this work discusses possible ways to accelerate separations and, in the same time perform these separations at the same level of efficiency as they have today. It seems possible to pack columns with smaller particles, probably down to 1μm and operate them with current vHPLC equipments for separations of biochemicals. Analyses of low molecular weight compounds will require new micro-HPLC systems able to operate 1mm I.D. columns at pressures up to 5kbar, which would eliminate the heat friction problems, and providing extra-column band broadening contributions smaller than 0.1μL2. Alternatively, a new generation of vHPLC systems with minimal extra-column contributions of less than 0.5μL2 could run 2.1mm I.D. columns if these latter were to be packed with high heat conductivity materials such as core–shell particles made with an alumina or gold core.

The effect of hydrothermal treatment on column performance for monolithic silica capillary columns

Monolithic silica capillary columns with i.d. 100 μm and monolithic silica rods were prepared with tetramethoxysilane (TMOS) or a mixture of TMOS and metyltrimethoxysilane (MTMS) using different hydrothermal treatments at T = 80 °C or 120 °C. Nitrogen physisorption was applied for the pore characterization of the rods and inverse size exclusion chromatography (ISEC) for that of the capillary columns. Using nitrogen physisorption, it was shown change of pore size and surface area corresponds to that of hydrothermal treatment and silica precursor. The results from ISEC agreed well with those from nitrogen physisorption regarding the pore size distribution (PSD). In addition, the retention factors for hexylbenzene with the ODS-modified capillary columns in methanol/water = 80/20 at T = 30 °C could also support the results from nitrogen physisorption. Furthermore, column efficiency for the columns was evaluated with alkylbenzenes and three kinds of peptides, leucine-enkephalin, angiotensin II, and insulin. Column efficiency for alkylbenzenes was similar independently of the hydrothermal treatment at T = 120 °C. Even for TMOS columns, there was no significant difference in column efficiency for the peptides despite the difference in hydrothermal treatment. In contrast, for hybrid columns, it was possible to confirm the effect on hydrothermal treatment at T = 120 °C resulting in a different column efficiency, especially for insulin. This difference supports the results from both nitrogen physisorption and ISEC, showing the presence of more small pores of ca. 3–6 nm for a hybrid silica without hydrothermal treatment at T = 120 °C. Consequently, the results suggest that hydrothermal treatment for a hybrid column with higher temperature or longer time is necessary, compared to that for a TMOS column, to provide higher column efficiency with increase in molecular size of solute.

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile–water (80/20, v/v) mobile phase at 40 °C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K F about 2.4 × 10 −14 m 2 (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000–120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates ( N) is greater than 50,000.

A simple cladding process to apply monolithic silica rods in high performance liquid chromatography

A simple cladding procedure was developed to apply monolithic silica rods in chromatography. This was used to evaluate the performances of new monolithic silica phases synthesised using a triblock copolymer as a phase separation inducer. The cladding procedure is found efficient after several tests, including scanning electron microscopy and mercury porosimetry, and the obtained columns present a relatively good efficiency in adsorption chromatography (H min is about 50 μm) as compared to other lab-scale developed columns.

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography

In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns.

Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption

Native and n-alkyl-bonded ( n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 μm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes.

Simple Liquid Chromatography Using Monolithic Silica Rod and Capillary

Simple liquid chromatography by capillary action using a monolithic silica rod and a capillary as the separation media instead of a thin-layer plate in conventional thin-layer chromatography (TLC) was examined. Possibilities of this chromatography were demonstrated by the separation of some organic compounds. Monolithic silica rod and capillary are very handy and useful as separation media for simple open liquid chromatography.

Determination of furosemide in plasma and urine using monolithic silica rod liquid chromatography

In the present study we developed a fast and reliable HPLC assay for the determination of the loop diuretic furosemide in plasma and urine, using a Chromolith ® RP 18e (100 mm × 4.6 mm) monolithic silica rod HPLC column. After liquid–liquid extraction with diethylether, plasma or urine samples were separated with a gradient consisting of solvent A (20% acetonitrile) and solvent B (80% acetonitrile), both in 0.25% acetic acid. The flow rate was 3.5 ml/min and the effluent was monitored by fluorescence with excitation at 230 nm and emission at 410 nm. The retention times for the internal standard (naproxen) and for furosemide were 2.1 and 3.7 min, respectively, and total run time was 8 min. The calibration curves were linear between 7.8 and 1000 ng/ml, and within-assay and between-assay coefficients of variation were <6.5% and <10%, respectively. The proposed assay for furosemide in plasma and urine using monolithic silica rod chromatography is fast, sensitive, and reliable, and, thus, well suited for pharmacokinetic studies.

Fast and reliable determination of the antifungal drug voriconazole in plasma using monolithic silica rod liquid chromatography

In the present study, we developed a fast and reliable HPLC assay for the determination of the new triazole antifungal agent voriconazole in plasma, using a Chromolith RP 18e (100 mm x 4.6 mm) monolithic silica rod HPLC column. After liquid-liquid extraction, plasma samples were separated with a mobile phase consisting of ammoniumdihydrogencarbonate buffer (pH 5.8)-acetonitrile-tetrahydrofuran (72:25:3) at a flow-rate of 3.5 mL/min and UV detection at 255 nm. The retention times for voriconazole and internal standard (UK-115794) were 2.3 and 2.7 min, respectively, and total run time was 4 min. The calibration curves were linear between 0.05 and 10 microg/mL, and within-assay and between-assay coefficients of variation were <4%. The proposed assay for voriconazole in plasma is fast, sensitive and reliable, and, thus, well suited for routine therapeutic monitoring of patients and for pharmacokinetic studies. It can be predicted that the use of monolithic silica rod chromatography will substantially shorten the turn-around time in the therapeutic drug monitoring laboratory.

In-Gel Digestion of Proteins Using a Solid-Phase Extraction Microplate

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.

Comparison of Selected Stationary Phases for Determination of Vancomycin and Ciprofloxacin Using Buffered Mobile Phases, With and Without Triethylamine

In our study we measured the retention factors, the theoretical plate numbers, and the symmetry factors in the case of phosphate buffer and triethylamine containing phosphate buffer, in order to get some information about the surface characteristic of tested columns. Three solutes (N,N‐dimethyl‐aniline, Ciprofloxacin, and Vancomycin) and 7 stationary phases (Nucleosil® 100‐C‐8, LiChrosorb® RP‐select B, LiChrospher® RP‐18, Purospher® RP‐18e, Prontosil® 120‐5‐C18‐AQ, Symmetry® C‐18, and Chromolith® SpeedROD RP‐18e) were used in this study. One of the solutes, N,N‐dimethyl‐aniline (121 g/mol) is a basic, low molecular weight compound. This is widely used for characterization of silanol activity of chemically bonded phases. The other two, Ciprofloxacin (331 g/mol) and Vancomycin (1449 g/mol), contain both basic and acidic groups, and Vancomycin has a high molecular weight. These latter two are real life samples and we tried to find a correlation between results of test compounds and these widely used antibiotics. For this purpose, we selected widely different columns: end‐capped phases, non end‐capped phases with high and low metal content, and last but not least, a recently developed monolithic silica column. According to selection criteria (N/m > 20,000; 0.8 < A S < 1.8), we evaluated the effect of masking agent (triethylamine) and the role of organic solvents (methanol and acetonitrile). It was found that the Symmetry C‐18 was the best in the case of Ciprofloxacin and N,N‐dimethyl‐aniline, and the Chromolith SpeedROD RP‐18e monolithic silica rod in the case of high molecular weight Vancomycin in these experiments. In most of the cases the LiChrosorb RP‐select B had to be excluded. Based on selection criteria, the eluent composition and the structure and size of tested solutes strongly influenced the applicability of other columns. Influence of TEA on theoretical plate number and symmetry factor was very high in the case of these columns.

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns.

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.

Monolithic silica rod liquid chromatography with ultraviolet or fluorescence detection for metabolite analysis of cytochrome P450 marker reactions

In vitro cytochrome P450 assays are used in metabolism studies in support of early phases of drug discovery to investigate, e.g., metabolic stability, enzyme inhibition and induction by new chemical entities. LC–UV and LC–fluorescence are traditional analytical tools in support of such studies. However, these tools typically comprise different methods of relatively low throughput for the various metabolites of probe reactions. In recent years, LC–MS methods have been developed to increase throughput. Increased throughput can also be achieved by means of modern chromatographic tools in combination with UV and fluorescence detection. This approach is especially suitable when cytochrome P450 isoforms are investigated by means of single probe incubations. Here, an LC–UV/fluorescence system based on a monolithic porous silica column is described for the analysis of metabolites of nine cytochrome P450 marker reactions [phenacetin to paracetamol (CYP1A2), coumarin to 7-hydroxycoumarin (CYP2A6), paclitaxel to 6α-hydroxypaclitaxel (CYP2C8), diclofenac to 4-hydroxydiclofenac (CYP2C9), mephenytoin to 4-hydroxymephenytoin (CYP2C19), bufuralol to 1-hydroxybufuralol (CYP2D6), chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1), midazolam to 1-hydroxymidazolam (CYP3A4), and testosteron to 6β-hydroxytestosteron (CYP3A4)]. While offering sensitivities and linear ranges comparable to previously reported methods, the set-up described here provides ease of use and increased throughput with maximum cycle times of 4.5 min.

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry.

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.