Toxicokinetic evaluation of atrasentan in mice utilizing serial microsampling: validation and sample analysis in GLP study.

BACKGROUND: A semi-automated 96-well protein precipitation followed by HPLC-MS/MS method for the determination of atrasentan (2R-[4-methoxyphenyl]-4S-[1,3-benzodioxol-5-yl]-1-[N,N-di-(N-butyl)-aminocarbonyl-methyl]-pyrrolidine-3R-carboxylic acid) in mouse whole blood was developed, validated and utilized in GLP toxicokinetic evaluations. Six 40-µl whole blood samples were collected from a single mouse over the course of a 12 h blood collection window. To avoid sample volume losses, whole blood was selected as the matrix in place of the more typically used plasma. A 10-µl assay volume was used to ensure sufficient volumes are available for dilutions, repeats and incurred sample reanalysis. The samples (10-µl aliquot) were fortified with stable-labeled internal standard (d18-atrasentan) and lysed thoroughly prior to protein precipitation. The chromatographic separation was performed on a Zorbax(®) SB-C18 (50 x 2.1 mm; 5 µm) HPLC column with a mobile phase consisting of 25 mM ammonium acetate and 0.25% (v/v) acetic acid in 50/50 (v/v) acetonitrile/water. The MS measurement was conducted under positive ion mode using multiple-reaction monitoring of m/z 511→354 for analyte and 529→354 for stable-labeled internal standard. The peak area ratio (analyte:stable-labeled internal standard) was used to quantitate atrasentan. RESULTS: A dynamic range of 5-1400 ng/ml was established after validation. The challenges associated with a small-volume whole-blood assay involved anticoagulant overloading with commercial blood collection tubes, managing phospholipids to ensure a robust assay and automation. In-depth discussions are provided in this article. The validated method was then used for GLP toxicokinetic evaluations. To demonstrate the method reproducibility, approximately 10% of the incurred samples from the study were repeated in singlet. Excellent assay reproducibility was demonstrated where 100% of samples met incurred sample reanalysis acceptance criteria. CONCLUSION: Good quality exposure data were obtained from every serial sampled mouse in the study.

Implementing DBS methodology for the determination of Compound A in monkey blood: GLP method validation and investigation of the impact of blood spreading on performance.

BACKGROUND: This article describes validation work for analysis of an Abbott investigational drug (Compound A) in monkey whole blood with dried blood spots (DBS). The impact of DBS spotting volume on analyte concentration was investigated. RESULTS: The quantitation range was between 30.5 and 10,200 ng/ml. Accuracy and precision of quality controls, linearity of calibration curves, matrix effect, selectivity, dilution, recovery and multiple stabilities were evaluated in the validation, and all demonstrated acceptable results. Incurred sample reanalysis was performed with 57 out of 58 samples having a percentage difference (versus the mean value) less than 20%. A linear relationship between the spotting volume and the spot area was drawn. The influence of spotting volume on concentration was discussed. CONCLUSION: All validation results met good laboratory practice acceptance requirements. Radial spreading of blood on DBS cards can be a factor in DBS concentrations at smaller spotting volumes.

Effect of sample collection tubing type used in a clinical study on quantitation of pharmaceutical compounds in CSF by LC-MS/MS.

BACKGROUND: plasma/cerebrospinal fluid (CSF) ratio of compound K was determined to be lower than predicted during the conduct of clinical study. This triggered the evaluation of nonspecific binding of drug K and an additional ten compounds in CSF to collection tubing. Physiochemical properties such as pKa and log D values were correlated to the quantified results. RESULTS: mean recoveries of compound K quality controls sampled within 24 h, as compared with spiked quality control, were 12.9, 44.4 and 77.7% from silicone, Tygon and Pharmed collection tubing, respectively. Eight out of ten compounds showed significant loss into silicone and seven out of ten compounds showed no significant loss into Pharmed. CONCLUSION: silicone tubing is not recommended and Pharmed appears to be most appropriate. CSF sample collection tubing must be evaluated prior to starting clinical studies.

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.

Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC-MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC-MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 degrees C) temperature and the low temperature (4 degrees C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2-4 degrees C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS-->SSA to < or = 0.10% and the conversion of SSA-->SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL(-1) for both SS and SSA and a sample volume of 100 microL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100+/-20% of the original analysis results.

Strategies for Developing Sensitive and Automated LC-MS/MS Assays of a Pharmaceutical Compound and Its Metabolite from Whole Blood Matrix.

When compared with biological samples in other matrices (plasma, urine, etc.) that are typically seen in bioanalytical applications, whole blood samples present unique challenges in method development, because of the viscous nature of blood and complexity of its constituents. In this article, we have developed and validated a series of quantitative bioanalytical methods for the determination of a pharmaceutical compound, Compound A, and its phosphate metabolite from whole blood matrices using liquid chromatography tandem mass spectrometry. All methods employed a simple protein precipitation procedure that was automated in 96-well format. The methods were subjected to vigorous tests in precision, accuracy, matrix effect, reproducibility, and robustness. Monolithic chromatography was used to improve sample throughput in one of the methods. The results also demonstrated that proper sample preparation procedures, such as sample transfer and lysing of blood cells prior to the extraction, are key to reproducible results for pharmacokinetic parameter determination.

Quantitative Determination of ABT-925 in Human Plasma by On-Line SPE and LC-MS/MS: Validation and Sample Analysis in Phase II Studies.

A fully automated 96-well On-Line Solid Phase Extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometric (MS/MS) method for the determination of ABT-925 (2-{3-[4-(2-tert-Butyl-6-trifluoromethyl-pyrimidin-4-yl)-piperazin-1-yl)-propyl-sulfanyl}-3H-pyrimidin-4-one fumarate) in human plasma was developed, validated and utilized in Phase II clinical studies. 50 µL of plasma sample was fortified with internal standard (IS, d8-ABT-925) and extracted on-line with Cohesive Turbo Flow Cyclone P HTLC column. The chromatographic separation was performed on Aquasil C18 (3 µm 50 × 3 mm) HPLC column with a mobile phase consisting of 50/50/0.1 (v/v/v) ACN/H2O/formic acid. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 457.4 → 329.4 for analyte and m/z 465.5 → 337.5 for IS.The peak area ratio (analyte/IS) was used to quantitate ABT-925. A dynamic range of 0.0102 µg/mL to 5.24 µg/mL was established after the validation. The validated method was then used for two Phase II studies. To demonstrate the method reproducibility, approximately 10% of the incurred samples from one study were repeated in singlet. The repeated values were compared to the initial values. All repeated values agreed within ±15% of the mean values.

Rapid, simultaneous determination of lopinavir and ritonavir in human plasma by stacking protein precipitations and salting-out assisted liquid/liquid extraction, and ultrafast LC-MS/MS.

Lopinavir and ritonavir are co-formulated in Kaletra approved for the treatment of human immunodeficiency virus infection. A validated analytical method is mandatory for clinical development and therapeutic drug monitoring. Here we are reporting a method for rapid, simultaneous determination of lopinavir and ritonavir in human plasma with stacked protein precipitations and salting-out assisted extraction (SALLE), and ultrafast LC-MS/MS detection. With stacked protein precipitations and SALLE, the sample preparation for a 96-well plate can be completed within 20 min by an automated pipette. Due to the unique cleanliness of SALLE extracts post double protein precipitations, the extracts were injected into an ultrafast liquid chromatography and tandem mass spectrometry system (LC-MS/MS) after simple dilution. An Agilent Zobax Extend-C18 Rapid resolution HT column (1.8 microm, 2.1 mm x 30 mm) was used for the separation. A mixture of acetonitrile:water (55:45, v/v) with 0.1% formic acid was used as the mobile phase. LC ran for approximately 48 s at a flow rate of 0.5 mL min(-1), tandem mass spectrometric data collection started at 15 s and lasts for 30 s. The method was validated with reference to Industry Guidance for Bioanalytical Method Validation and then used for clinical samples. The method is ultrafast, and robust. Results of incurred samples demonstrated excellent method of reproducibility. This ultrafast analysis speed did not compromise with the data quality. To our knowledge, this is the fastest analytical method for simultaneous determination of lopinavir and ritonavir.

Highly sensitive LC-MS-MS analysis of a pharmaceutical compound in human plasma using monolithic phase-based on-line extraction.

Monolithic columns have been used in recent years for fast chromatographic separation due to their high permeability and low backpressure. We have explored the potential of monolithic material as sample preparation tool in bioanalytical applications. By taking advantage of monolithic columns' online concentration capability, we have developed a highly sensitive liquid chromatography-tandem mass spectrometry assay for quantitative determination of a pharmaceutical compound in human plasma. The assay was fully validated to satisfy the requirements of precision and accuracy, selectivity, matrix effect, and reproducibility. A linear dynamic range from 0.011 ng/mL to 12.3 ng/mL was established as the calibration standard. The percentage of bias for quality control samples was between -9.9% and -2.5%. Coefficient of variation, a measurement of precision, was within 9.9%. On-line extraction with monolithic support provided adequate sample cleanup and on-line concentration of the analyte. The assay exhibited good tolerance to matrix effect and has been applied successfully to a clinical study. The incurred sample analysis showed that original and repeat values were within +/-10.1% for all assay samples.

Salting-out assisted liquid/liquid extraction with acetonitrile: a new high throughput sample preparation technique for good laboratory practice bioanalysis using liquid chromatography-mass spectrometry.

Acetonitrile, an organic solvent miscible with aqueous phase, has seen thousands of publications in the literature as an efficient deproteinization reagent. The use of acetonitrile for liquid-liquid extraction (LLE), however, has seen very limited application due to its miscibility with aqueous phase. The interest in LLE with acetonitrile has been pursued and reported in the literature by significantly lowering the temperature of the mixture or increasing the salt concentration in the mixture of acetonitrile and aqueous phase, resulting in the separation of the acetonitrile phase from aqueous phase, as observed in conventional LLE. However, very limited application of these methods has been reported. The throughput was limited. In this report, we report a new sample preparation technique, salting-out assisted liquid-liquid extraction with acetonitrile, for high-throughput good laboratory practice sample analysis using LCMS, Two compounds from an approved drug, Kaletra, were used to demonstrate the extractability of drugs from human plasma matrix. Magnesium sulfate was used as the salting-out reagent. Extracts were diluted and then injected into a reversed phase LC-MS/MS system directly. One 96-well plate was extracted with this new approach to evaluate multiple parameters of a good laboratory practice analytical method. Results indicate that the method is rapid, reliable and suitable for regulated bioanalysis. With minimal modification, this approach has been used for high-throughput good laboratory practice analysis of a number of compounds under development at Abbott.

High-throughput salting-out assisted liquid/liquid extraction and ultrafast LC for same-day delivery of first-in-human bioanalytical data.

BACKGROUND: With the need of fast-paced drug development, rapid delivery of bioanalytical data becomes a trend. Here, we present a strategy to demonstrate same-day data delivery. RESULTS: A novel salting-out assisted liquid/liquid extraction (SALLE) with acetonitrile and a MS-friendly salt was used to extract analyte from the first-in-human study plasma samples and the extract was successfully injected into ultrafast chromatography. The strategic combination of SALLE and ultrafast chromatography minimizes the turnaround time and allows the same-day delivery of bioanalytical data. The time saving from both extraction and injection was translated to a fast delivery of bioanalytical data. CONCLUSION: The first-in-human pharmacokinetic data of an investigational new drug candidate was delivered in approximately 4.5 work h after receiving the samples of each dose group using high-throughput SALLE and ultrafast LC. Incurred sample reassay results proved uncompromised data quality with the high-speed bioanalysis.

Bioanalytical hydrophilic interaction chromatography: recent challenges, solutions and applications.

Hydrophilic interaction chromatography (HILIC) has, in recent years, been shown to be an important supplement to reversed-phase liquid chromatography for polar analytes. HILIC, in conjunction with tandem mass spectrometry (MS/MS), has been steadily gaining acceptance in the analysis of polar compounds from complex biological matrices. This hyphenated technique offers the advantages of improved sensitivity by employing high organic content in the mobile phase, shortened sample preparation time with direct injection of the organic-solvent extracts of biological samples and the potential for ultra-fast analysis because of low-column backpressure. This article reviews recent challenges presented by HILIC, advancements in the better understanding of retention characteristics of analytes with different mobile- and stationary-phase compositions and solutions to ion suppression and interference problems encountered in HILIC-MS/MS assays. Applications of HILIC-MS/MS are summarized, including those for pharmacokinetic studies, metabolic studies, therapeutic drug monitoring and clinical diagnostics.

Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt.

In previously reported applications of salting-out assisted liquid/liquid extraction (SALLE) with acetonitrile, only inorganic salts were evaluated and implemented as the salting-out reagents. A potential concern of the method for the subsequent LC-MS analysis of biological samples was that a portion of the added salt (typically of high concentration) might be extracted and affect the chromatography separation and ionization of chromatography effluents in a mass spectrometer. Here we report, for the first time, the use of a mass spectrometry friendly organic salt, ammonium acetate, as a salting-out reagent in SALLE with acetonitrile for the simultaneous quantitation of an Abbott investigational new drug ABT-869 and its hydrophilic metabolite in human plasma. The performance of SALLE with ammonium acetate was compared with that of a previously reported method with a conventional liquid/liquid extraction technique using a set of pooled incurred samples. The % differences of the measured concentrations for 24 samples from these two methods were found to be within acceptance criteria, demonstrating SALLE with ammonium acetate as a reliable sample preparation technique. The SALLE method is simple, fast (25 min/plate), easy for automation, free of drying down step, and environmentally friendly. SALLE with mass spectrometry friendly salts has been applied to regulated sample analysis of both hydrophilic and hydrophobic compounds. It is envisioned that SALLE with acetonitrile and ammonium acetate be a universal method for high throughput automated sample preparation for bioanalytical chemistry.

HPLC-MS/MS determination of a hardly soluble drug in human urine through drug-albumin binding assisted dissolution.

ABT-263 is under development for treatment of cancer. In order to support clinical trials, an analytical method for ABT-263 quantification in human urine became necessary. Due to the extremely poor solubility of ABT-263 in aqueous and most common organic solvents, a critical step was to dissolve the drug into urine matrix. Although other potential approaches could be used, addition of powder albumin was found to be the most advantageous. Albumin powder does not significantly alter urine sample volume (< or = 2.8%) and a range of albumin to urine sample volume ratios can be allowed for full recovery of drug and thus accurate quantification. The procedure is fairly simple and can potentially be a universal approach for compounds with low solubility in urine, but strong protein binding. The method has been validated to support clinical trials.

Simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its metabolite in urine using online monolithic phase-based extraction.

This article describes the use of monolithic material as extraction support in simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its hydroxylated metabolite in urinary matrix. DMSO was added to urine samples to aid the solubilization of analytes during storage and transfer. Samples were then diluted and injected onto an online extraction system for high-throughput analysis in an automated fashion. A linear range of 1.03-103 ng/mL was validated for the parent compound and 7.02-1400 ng/mL for the metabolite. The accuracy (%bias) at the lower LOQ (LLOQ) for the parent compound was -2.9% and the precision (%CV) at the LLOQ was 5.4%, while the accuracy at LLOQ for the metabolite was 6.3% and the precision at LLOQ was 5.5%. The results show that quantitative LC-MS/MS analysis by online extraction using the monolithic support is reproducible, sensitive, and accurate enough to satisfy bioanalytical testing requirements in a good laboratory practice (GLP) regulated environment. Monolithic phase based online extraction provided efficient sample cleanup, which was evidenced in matrix effect testing against multiple lots of urinary matrix. The method described within can be a generic approach for quantitative analysis of analytes with poor solubility in aqueous matrix.

A strategy for high-throughput analysis of levosimendan and its metabolites in human plasma samples using sequential negative and positive ionization liquid chromatography/tandem mass spectrometric detection.

Levosimendan (Simdax) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high-throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high-performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR-1855 and OR-1896. Here, we present a novel strategy for high-throughput analysis of levosimendan and its metabolites. A 96-well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200-50,000 pg/mL for levosimendan and approximately 500-130,000 pg/mL for both metabolites. The coefficient of determination (r2) for all analytes was greater than 0.9985. The intra-assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR-1855, and 0.4% to 4.9% for OR-1896. The inter-assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR-1855, and 1.4% to 3.4% for OR-1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, -1.4% to 2.6% for OR-1855, and -0.3% to 4.5% for OR-1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage.

LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparation.

As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 x 150 mm, 5 microm, 120 A) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 microL sample volume. The achieved r(2) coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between -4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between -8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was < or =4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies.

Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection.

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre-cut arteries within pre-defined masses. A high-throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between -4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were < or =5.3% for the quality controls in blood and < or =9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries.

Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS.

Liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages since its introduction to the pharmaceutical industry. This article reviews the most recent advances in sample preparation, separation, and the mass spectrometric aspects of high-throughput quantitative bioanalysis of drug and metabolites in biological matrices. Newly introduced techniques such as ultra-performance liquid chromatography with small particles (sub-2 microm) and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC-MS/MS. Sample preparation formatted to 96-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly impacting throughput. On-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both time and labor required to produce bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media including large particles for turbulent flow chromatography, restricted access materials (RAM), monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland systems. In the end, this paper also discusses recent studies of matrix effect in LC-MS/MS analysis and how to reduce/eliminate matrix effect in method development and validation.

Accurate determination of an immunosuppressant in stented swine tissues with LC-MS/MS.

During stent development, accurate monitoring of the drug concentration in animal tissues can provide critical information on how the drug is released into the circulation and the surrounding tissues. To establish the relationship between the drug concentration and the distance from the stent to the target tissue, a comprehensive strategy was developed for sample collection, sample homogenization and sample storage as well as sample analysis. This strategy was developed with the analytical chemists and animal surgical specialists working together as a team. The optimized sampling process was designed to yield a representative sample, appropriately located and of an appropriate size. The sampling process was also designed to eliminate the potential for carryover and cross-contamination. During sample processing, the analyte solution was spiked into blank tissues using a sharp needle and a gas-tight syringe to prepare tissue quality control samples. These tissue quality controls were then used to evaluate the stability of the drug in solid tissue and homogenate, the homogenization carryover, the cross-contamination and the recovery of the drug during method validation and to monitor the overall process of drug analysis of the swine tissues. This thorough strategy has been applied to the accurate determination of zotarolimus in swine tissues for regulated toxicology studies. The entire process was controlled, including precise tissue sampling, compound-based tissue homogenization, method validation, and the application of the method to regulated toxicokinetics studies. The results demonstrate that analytical chemistry concepts can be successfully integrated into toxicokinetics studies in order to collect precise samples and obtain meaningful results. The strategy can be applied to similar toxicokinetics studies of locally administrated drugs in tissues.

A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody.

The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.