Solid-phase microextraction in bioanalysis: New devices and directions

The primary objective of this review is to discuss recent technological developments in the field of solid-phase microextraction that have enhanced the utility of this sample preparation technique in the field of bioanalysis. These developments include introduction of various new biocompatible coating phases suitable for bioanalysis, such as commercial prototype in vivo SPME devices, as well as the development of sampling interfaces that extend the use of this methodology to small animals such as mice. These new devices permit application of in vivo SPME to a variety of analyses, including pharmacokinetics, bioaccumulation and metabolomics studies, with good temporal and spatial resolution. New calibration approaches have also been introduced to facilitate in vivo studies and provide fast and quantitative results without the need to achieve equilibrium. In combination with the drastic improvement in the analytical sensitivity of modern liquid chromatography–tandem mass spectrometry instrumentation, full potential of in vivo SPME as a sample preparation tool in life sciences can finally be explored. From the instrumentation perspective, SPME was successfully automated in 96-well format for the first time. This opens up new opportunities for high-throughput applications (>1000 samples/day) such as for the determination of unbound and total drug concentrations in complex matrices such as whole blood with no need for sample pretreatment, studies of distribution of drugs in various compartments and/or determination of plasma protein binding and other ligand–receptor binding studies, and this review will summarize the progress in this research area to date.     

An integrated bioanalytical platform for supporting high-throughput serum protein binding screening

Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.     

Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background

Protein expression alterations unrelated to an investigated phenotype are accumulated in most cell line models during establishment. Performing a whole proteome screening of lymphoma cell lines, we established a method to reduce the influence of protein expression unrelated to the distinct investigated phenotype. In 2-D PAGE, the comprehensive analysis of a large number of protein spots would be simplified by pooling cell line samples of the investigated phenotype. Applying this pooling approach, unrelated alterations of single samples are 'muted' by dilution. Analysing two different lymphoma subtypes (follicular and mantle cell lymphoma) by this method, spots originating in only single cell lines were reduced by 72% (650/900), whereas even modestly altered expression of protein spots detected in all lines were reliably detected in the pooled protein gels. We conclude that our pooling approach is a preferable approach to reliably detect a common protein expression pattern and may even allow proteomic analysis of clinical samples with limited amounts of sample material, even with minimal cell numbers as low as 1 x 10(6).     

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Simple and Rapid Hollow Fiber Liquid Phase Microextraction Followed by High Performance Liquid Chromatography Method for Determination of Drug-protein Binding

A method was established using hollow fiber-liquid phase microextraction(HF-LPME) followed by high performance liquid chromatography(HPLC) to determine the concentration of the free(unbound) drug in the solution of the drug and protein. Measurements of drug-protein binding ratios and free drug concentrations were then analyzed with the Klotz equation to determine the equilibrium binding constant and number of binding sites for drug-protein interaction. The optimized method allows one to perform the efficient extraction and separation of free drug from protein-bound drug, protein, and other interfering substances. This approach was used to characterize the binding of the anticholinergic drugs atropine sulfate and scopolamine hydrobromide to proteins in human plasma and bovine serum albumin(BSA). The results demonstrate the utility of HF-LPME method for measuring free drug concentrations in protein-drug mixtures and determining the protein binding parameters of a pharmacologically important class of drugs.     

Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis

Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to HSA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 microM (+/-)AL was equilibrated with 300 microM HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and KS-AL, were 9910-11200 and 90200-104000 M(-1), respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site I). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.     

高效迎头分析法测定药物-人血清白蛋白混合液中游离药物浓度

 用高效迎头分析法 (HPFA)测定了药物 人血清白蛋白 (HSA)混合液中游离药物的浓度。样品溶液不经任何处理直接进样到装有内表面反相固定相的色谱柱中 ,用 67mmol/L磷酸盐缓冲液 ( pH 7 4 ,I =0 17mol/L)作流动相。当进样体积足够大时 ,游离药物以平顶峰的形式被洗脱出来 ,平顶峰区域洗脱液中的药物浓度等于样品溶液中游离药物的浓度。收集平顶峰区域的洗脱液 ,然后将一定体积的洗脱液注入到反相色谱柱中 ,测定游离药物的浓度。用该法测定酮基布洛芬 HSA和头孢哌酮 HSA两种混合液中游离药物的浓度。     

Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.     

A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.     

Single run measurements of drug-protein binding by high-performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.     

LC–MS bioanalysis of intact proteins and peptides

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC–MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC–MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC–MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.     

Enantioselective binding analysis of verapamil to plasma lipoproteins by capillary electrophoresis-frontal analysis

Capillary electrophoresis coupled with frontal analysis was applied to the study of enantioselective binding of verapamil (VER) to plasma lipoproteins. The drug-lipoprotein mixed solution, which had been in the binding equilibrium, was hydrodynamically introduced into a non-coated fused-silica capillary. Since VER is positively charged in the neutral run buffer (pH 7.4), the unbound VER enantiomers migrated toward the cathodic end much faster than negatively charged lipoproteins and their bound forms. Once unbound VER migrated apart from lipoprotein, the bound VER was quickly released from the protein to maintain the binding equilibrium. Thus, VER migrated as a zone through the capillary and gave a trapezoidal peak with a plateau region on the electropherogram. The VER concentration in this plateau region was equal to the unbound VER concentration in the initial sample solution. It was found that the bindings of VER to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL were not site-specific and not enantioselective. Partition-like binding to lipid part of these lipoproteins seemed to be dominant. The total binding affinities of LDL to VER were about seven-times stronger than those of HDL, and the oxidation of LDL by copper ion enhanced the binding affinities significantly.     

Pulse gradient, large-volume injection, high-throughput ultra-performance liquid chromatographic/tandem mass spectrometry bioanalysis for measurement of plasma amrubicin and its metabolite amrubicinol

In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of amrubicin (an anthracycline anti-cancer agent) and its active metabolite, amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with formic acid was associated with improvement in the lower limit of quantification. These favorable effects were electrolyte concentration-dependent. In order to maximize assay throughput, we used methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.     

A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies

For the development of human antibody Fc (fraction crystallizable) region-containing therapeutic protein candidates, which can be either monoclonal antibodies (mAbs) or pharmacologically active proteins/peptides fused to the Fc region of human Immunoglobulin G (IgG), reliable quantification of these proteins in animal pharmacokinetic study plasma samples is critical. LC-MS/MS has emerged as a promising assay platform for this purpose. LC-MS/MS assays used for bioanalysis of human antibody Fc region-containing therapeutic protein candidates frequently rely upon quantification of a 'signature' surrogate peptide whose sequence is unique to the protein analyte of interest. One drawback of the signature peptide approach is that a new LC-MS/MS assay must be developed for each new human Fc region-containing therapeutic protein. To address this issue, we propose an alternative 'universal surrogate peptide' approach for the quantification of human antibody Fc region-containing therapeutic protein candidates in plasma samples from all nonclinical species. A single surrogate tryptic peptide was identified in the Fc region of most human antibody Fc-containing therapeutic protein candidates. An LC-MS-MS method based upon this peptide was shown to be capable of supporting bioanalysis of a diversity of human Fc region-containing therapeutic protein candidates in plasma samples of all commonly used animal species.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening

Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K_d determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K_d calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.     

Development of a Rapid and Low Cost Method for Measuring Plasma Protein Binding

In drug discovery and development, it is very important to investigate the plasma protein binding (PPB) of a drug to better understand its in vivo fate. In this study, a rapid and low-cost solid-phase extraction (SPE) method was developed for determining the PPB. With this method, the total protein recovery of a blank human plasma sample was 83.7 %. The unbound drug was easily adsorbed by an ODS C₁₈ SPE column, and the recovery of three known drugs was more than 90 %. Their PPBs obtained by the SPE were identical to the value reported by conventional techniques. In addition, more than 90 % of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), which is a novel all-trans retinoic acid derivative (ATRA), was bound to human plasma protein as determined by SPE, and this value was comparable with that obtained by our previously described gel filtration-based method. Considering its versatility, speed of separation, and low cost, SPE is a rapid and economical method for measuring PPB.

     

Molecular modeling approaches for the prediction of the nonspecific binding of drugs to hepatic microsomes

Molecular modeling approaches for the prediction of the nonspecific binding of drugs to hepatic microsomes were examined using a published database of 56 compounds. Models generated were evaluated using an independent test set of 13 compounds. A pharmacophore approach identified structural features of drugs associated with nonspecific binding. A side-chain amino group and complementary hydrophobic domain were the principal features noted. The use of shape overlays, based on the pharmacophore, in conjunction with a chemical force field in the program ROCS, yielded discrimination between molecules classified as strong binders (experimental fraction unbound in microsomes<0.50) and those with a lower degree of binding (experimental fraction unbound in microsomes>0.50). In the initial data set of 56 molecules, 18 were classified as strong binders (on the basis of the above criteria), and all of those were recovered in the top 22 molecular hits from ROCS. Additionally, computationally generated values of log P were shown to provide a reasonable estimate of the fraction unbound in microsomes, providing the compounds were in their basic form at physiological pH.     

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.