Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.     

Rapid drug metabolite profiling using fast liquid chromatography, automated multiple-stage mass spectrometry and receptor-binding

Rapid drug metabolite profiling can be achieved using fast chromatographic separation and fast mass spectrometric scanning without compromising the separation efficiency. Fast chromatographic separations of drug and its metabolites can be achieved by eluting from a short narrow-bore guard cartridge column (20 x 2 mm I.D., 3 microns BDS Hypersil C8) at flow-rate of 1.0 ml/min and with a gradient volume greater than 90 column volumes. The need for chromatographic separation is important for automated data dependent multiple-stage mass spectrometry (MSn) experimentation. The total analysis time of 8 min permits profiling of metabolites in a 96-well plate in 13 h. The narrow chromatographic peaks resulting from the high flow-rate require the use of a mass spectrometer capable of fast scan speed due to the need to perform multiple MS experiments within the same chromatographic analysis. A method has been developed for screening potentially biologically active in vitro microsomal metabolites by affinity binding with a receptor. After separation by centrifugal ultrafiltration, the bound ligands are released and characterized by LC-MS. In vitro microsomal metabolites of tamoxifen, raloxifene and adatanserin were screened for potential biological activity using this method. The in vitro metabolites of tamoxifen captured by the receptor include N-demethyltamoxifen and three species of hydroxytamoxifen; these data are consistent with those from a conventional binding study and bioassay. In addition, both hydroxyraloxifene and dihydroxyraloxifene are also recognized by the receptor. The specificity of the molecular recognition process is illustrated by the absence of binding with control microsomal incubate and with adatanserin and its metabolites. Therefore, active metabolites can be rapidly profiled by fast LC, automated MSn, and receptor binding. This information can be obtained quickly and can add value to the drug discovery process.     

Liquid chromatography/tandem mass spectrometric quantification with metabolite screening as a strategy to enhance the early drug discovery process

Throughput for early discovery drug metabolism studies can be increased with the concomitant acquisition of metabolite screening information and quantitative analysis using ultra-fast gradient chromatographic methods. Typical ultra-fast high-performance liquid chromatography (HPLC) parameters used during early discovery pharmacokinetic (PK) studies, for example, employ full-linear gradients over 1-2 min at very high flow rates (1.5-2 mL/min) on very short HPLC columns (2 x 20 mm). These conditions increase sample throughput by reducing analytical run time without sacrificing chromatographic integrity and may be used to analyze samples generated from a variety of in vitro and in vivo studies. This approach allows acquisition of more information about a lead candidate while maintaining rapid analytical turn-around time. Some examples of this approach are discussed in further detail.     

High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50:50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs.     

Direct simultaneous analysis of plasma samples for a drug discovery compound and its hydroxyl metabolite using mixed-function column liquid chromatography-tandem mass spectrometry

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min(-1). When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5-2500 ng ml(-1) for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were > 90% (RSD < 6%). The total analysis time was < 8 min per sample. The analytical results obtained by the proposed direct plasma injection method were in good agreement with those obtained by the conventional LC-MS-MS method.     

Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for Cinchona alkaloid separations.     

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.     

Efficient method development strategy for challenging separation of pharmaceutical molecules using advanced chromatographic technologies

In this paper, we describe a strategy that can be used to efficiently develop a high-performance liquid chromatography (HPLC) separation of challenging pharmaceutical molecules. This strategy involves use of advanced chromatographic technologies, such as a computer-assisted chromatographic method development tool (ChromSword) and an automated column switching system (LC Spiderling). This process significantly enhances the probability of achieving adequate separations and can be a large time saver for bench analytical scientists. In our study, the ChromSword was used for mobile phase screening and separation optimization, and the LC Spiderling was used to identify the most appropriate HPLC columns. For proof of concept, the analytes employed in this study are the structural epimers betamethylepoxide and alphamethylepoxide (also known as 16-beta methyl epoxide and 16-alpha methyl epoxide). Both of these compounds are used in the synthesis of various active pharmaceutical ingredients that are part of the steroid pharmaceutical products. While these molecules are relatively large in size and contain various polar functional groups and non-polar cyclic carbon chains, their structures differ only in the orientation of one methyl group. To our knowledge, there is no reported HPLC separation of these two molecules. A simple gradient method was quickly developed on a 5 cm YMC Hydrosphere C(18) column that separated betamethylepoxide and alphamethylepoxide in 10 min with a resolution factor of 3.0. This high resolution provided a true baseline separation even when the concentration ratio between these two epimers was 10,000:1. Although outside of the scope of this paper, stability-indicating assay and impurity profile methods for betamethylepoxide and for alphamethylepoxide have also been developed by our group based on a similar method development strategy.     

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 microm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5-30 min, and even more rapid (1-2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high-performance liquid chromatography with diode-array detection.

The need for a rapid, sensitive and reliable analytical method for cyanobacterial toxins, microcystins, has been emphasized by the awareness of toxic cyanobacteria as a human-health risk through drinking water. A new high-performance liquid chromatographic method with column switching was developed for the determination of microcystin-LR, -RR and -YR from water samples without pre-purification. The filtered water sample was passed through a Zorbax CN precolumn at a flow-rate of 3 ml/min for on-line trace enrichment. After valve switching, concentrated analytes were eluted in back-flush mode and separated on a Luna C18 column with a gradient of acetonitrile -20 mM phosphate buffer (pH 2.5). The method showed excellent precision, accuracy and speed with detection limits of 0.02 microgram/ml from 100 ml of surface water. The total analysis time per sample was about 90 min. This method improves reliability, sensitivity and sample throughput, and shortens the analysis time compared to analysis methods using off-line solid-phase extraction.     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Implementation of a rapid and automated high performance liquid chromatography method development strategy for pharmaceutical drug candidates

A comprehensive, fully automated strategy is demonstrated for HPLC-UV chromatographic method development using ChromSword optimization software. The strategy involves: (1) the automated screening of various column and mobile phase combinations, (2) rational selection of the best starting conditions; and (3) subsequent automated method development to generate optimized separation methods. Pharmaceutical compounds were applied to solve problematic drug impurity separations. ChromSword software automates the screening, optimization, and documentation steps thus reducing the method development time. The strategy was compared to a manual method development approach showing the automated method strategy affords better selectivity in a shorter time.     

High-efficiency liquid chromatography–mass spectrometry separations with 50 mm, 250 mm,and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests

In this study, high-efficiency LC–MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1 m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50 mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50 mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n_c = 370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1 m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.     

Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles

The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of an fast analysis method for the most relevant soy isoflavones. A step-by-step strategy was used to optimize temperature (25-50 °C), flow rate (1.2-2.7 mL/min), mobile phase composition and equilibration time (1-5 min). Optimized conditions provided a method for the separation of all isoflavones in less than 5.8 min and total analysis time (sample-to-sample) of 11.5 min. Evaluation of chromatographic performance revealed excellent reproducibility, resolution, selectivity, peak symmetry and low limits of detection and quantification levels. The use of a fused-core column allows highly efficient, sensitive, accurate and reproducible determination of isoflavones with an outstanding sample throughout and resolution. The developed method was validated with different soy samples with a total isoflavone concentration ranging from 1941.53 to 2460.84 μg g⁻¹ with the predominant isoflavones being isoflavone glucosides and malonyl derivatives.     

HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

Abstract

A sensitive high performance liquid chromatographic method for the simultaneous determination of 6-thiouric acid and 6-mercaptopurine in serum is described. Our intent was to develop a procedure that could be used for pharmacokinetic studies and therapeutic drug monitoring in organ transplant patients taking azathioprine. Serum samples were precipitated with acetonitrile containing 6-n-propyl-2-thiouracil as the internal standard. The chromatographic separation was performed with an octadecylsilane column and gradient solvent system consisting of acetonitrile and 0.01 M sodium dihydrogen phosphate, pH 6.1. An initial acetonitrile concentration of 1% was used to elute 6-thiouric acid but was increased to 16% to recover the 6-mercaptopurine and internal standard. The flow rate was increased from 1.3 ml/min to 1.5 ml/min during the analysis. The column effluent was monitored at 353 nm and 323 nm for detection of 6-thiouric acid and 6-mercaptopurine, respectively. Statistical analysis of standard curve data showed good intra- and inter-day accuracy, precision and reproducibility throughout a concentration range of 10–2500 ng for 6-thiouric acid and 10–500 ng for 6-mercaptopurine/ml of serum. The method has been applied to the quantification of 6-thiouric acid and 6-mercaptopurine in serum from two kidney allograft recipients.     

Automated two-dimensional liquid chromatographic system for mapping proteins in highly complex mixtures.

An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.     

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320min) and different column lengths (0.25, 1, 2 and 4m) all originating from a single 4m long column. Peak capacities reaching values up to n=10(3) were measured in case of the 4m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4m long column possessed a structural homogeneity over its entire length.     

Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery

High-throughput and performance analysis and purification of enantiomers are important parts of drug discovery and provide high-quality compounds for pharmacological testing. We have previously reported two parts describing chiral chromatographic screens using normal-phase (NPLC) and reversed-phase (RPLC) liquid chromatography, in order to cope with increasing numbers of new compounds generated by chemistry programs. We present in this part the development and implementation of a third faster screen using supercritical fluid chromatography (SFC) to maximize chance in achieving rapid enantiomer resolution of large numbers of compounds in a minimum of time. The SFC screen utilizes a narrow combination of only four columns (Chirlapak AD and AS, and Chiralcel OD and OJ) and two solvent modifiers (methanol and isopropanol). A modifier and column-switching setup was employed to allow the entire screening process to be serially run in the order AD> OD > OJ > AS and methanol > isopropanol, so that the screening for a given molecule can be stopped when separation is achieved. The switching system was fully automated for unattended operation of multiple compounds. An optimization procedure was also defined, which can be performed if needed for unsuccessful separations in the screening step. The chiral SFC strategy proved its performance and robustness in resolution of hundreds proprietary chiral molecules generated by drug discovery programs, with a success rate exceeding 95%. In addition, the generic capability of the strategy was evaluated by applying the screen and optimization methodology to a test set comprising 40 marketed drugs differing from proprietary compounds in terms of chemical diversity, revealing a similar high success rate of 98%. Chiral separations developed at the analytical scale work easily and equally well at the semi-preparative level, as illustrated with an example. The SFC screen allows resolution of compounds that were partially separated by NPLC or not separated at all by RPLC, demonstrating the utility of implementing complementary chromatographic techniques. The SFC screen is currently an integral part of our analytical support to discovery chemical programs and is considered the first try for chiral separations of new compounds, because it offers a higher success rate, performance and throughput.     

Development and Validation of a Stability-Indicating UPLC Method for the Determination of Hexoprenaline in Injectable Dosage Form Using AQbD Principles

A novel and efficient stability-indicating, reverse phase ultra-performance liquid chromatographic (UPLC^®) analytical method was developed and validated for the determination of hexoprenaline in an injectable dosage form. The development of the method was performed using analytical quality by design (AQbD) principles, which are aligned with the future requirements from the regulatory agencies using AQbD principles. The method was developed by assessing the impact of ion pairing, the chromatographic column, pH and gradient elution. The development was achieved with a Waters Acquity HSS T3 (50 × 2.1 mm i.d., 1.8 µm) column at ambient temperature, using sodium dihydrogen phosphate 5 mM + octane-1-sulphonic acid sodium salt 10 mM buffer pH 3.0 (Solution A) and acetonitrile (Solution B) as mobile phases in gradient elution (t = 0 min, 5% B; t = 1 min, 5% B; t = 5 min, 50% B; t = 7 min, 5% B; t = 10 min, 5% B) at a flow rate of 0.5 mL/min and UV detection of 280 nm. The linearity was proven for hexoprenaline over a concentration range of 3.50–6.50 µg/mL (R² = 0.9998). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability and injectable dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with enhanced knowledge, which can eventually lead to a reduced regulatory risk, high quality data and lower operational costs.