LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics.

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.

Microdialysis sampling of the isothiazolone, PD-161374, and its thiol and disulfide metabolites.

A method based on microdialysis sampling combined with high-performance liquid chromatography (HPLC) has been developed for monitoring the anti-HIV agent PD-161374 (isothiazolone) and its thiol and disulfide metabolites in blood. It was demonstrated that unlike blood withdraw and extraction, microdialysis sampling can preserve the distribution among the isothiazolone and its metabolites in blood. The use of a narrow-bore HPLC system, combined with the relatively high probe extraction efficiency (approximately 50%) from the flexible probe design in this work, allows the direct and quantitative determination of the drug and its major metabolites at submicromolar level.

Development and validation of a semi-automated method for L-dopa and dopamine in rat plasma using electrospray LC/MS/MS.

A semi-automated alumina-based extraction method for the determination of L-dopa and dopamine in plasma using liquid chromatography mass spectrometry was validated. The method exploited the use of a Tomtec Quadra 96 liquid handing robot to expedite aluminum oxide extraction for sample clean up. Two 96-well sample plates can be processed in less than 2 h and extracts, collected in a 96-well plate format, can be directly injected onto the ESI/LC/MS/MS instrumentation. Chromatographic separation of the analytes was performed on a reverse-phase ODS column (TosoHaas ODS-80) with a mobile phase of acetonitrile/0.1% formic acid (5/95 v/v) at a flow rate of 0.22 ml/min. Analytes were detected by a triple-quadruple mass spectrometer equipped with an electrospray ionization source (ESI). Recoveries were evaluated for a number of pH modifiers and elution solvents. Under optimized conditions, the mean recoveries of L-dopa and dopamine were 56 and 67%, respectively. Intra-run and inter-run precision, calculated as percent relative standard deviation of replicate quality controls, was in the range of 1.45-10.8% for both L-dopa and dopamine. Intra-run and inter-run accuracy, calculated as percent error, was in the range -2.5 to 6.69% for both analytes. The limit of quantitaiton was 2.5 ng/ml for both L-dopa and dopamine when 100 microl of plasma was extracted. The method is simple, rapid, accurate and suitable for the quantification of L-dopa and dopamine in plasma or other biological fluid samples from clinical, preclinical, or pharmacological studies.

Quantitative method for biomarkers of collagen degradation using liquid chromatography tandem mass spectrometry.

Preclinical efficacy testing commonly involves studies that require considerable resources and time. One valuable tool in this endeavor is the characterization of relevant biomarkers. A method has been developed for the simultaneous determination of collagen biomarker candidates as an instrument in screening compounds for efficacy. Two potential candidates, the 3-hydroxypyridinium crosslinks pyridinoline and deoxypyridinoline, were selected for analysis in collagen degradation models. Tissue or urine samples were collected, prepared, and quantitated for the biomarkers using spiked calibration curves and liquid chromatography tandem mass spectrometry. The development of a quick and simple assay method would allow us to increase the chances for success in efficacy screening by eliminating compounds with poor biomarker profiles. The method proposed here appears to be more selective, convenient, precise (generally <10% RSD), accurate (generally <10% RE), and sensitive relative to previously established methodology.

Integrated sample collection and handling for drug discovery bioanalysis.

An integrated sample handling process for drug discovery bioanalysis is described. The streamlining of study design, sample collection and automatic bioanalytical sample processing is demonstrated. Specific details for the entire procedure regarding the time saved, ease of automation and integration are defined. Details of sample handling involved a sample collection map, sample collection formatting and volume, dilution schemes for high concentration samples, choice of biological fluid and evaluating the capabilities of two liquid-handling workstations. Numerous comparisons were conducted between the new approaches and the conventional sample handling approaches. The precision and accuracy obtained from the new integrated sample handling process were comparable to those obtained from a conventional approach, as were pharmacokinetic profiles and parameters. This new sampling process greatly improved the efficiency of drug discovery bioanalysis. The integration of pre-clinical protocol design, sample collection and bioanalysis processes was also achieved.

Automating solid-phase extraction: current aspects and future prospects.

This paper reviews current trends and techniques in automated solid-phase extraction. The area has shown a dramatic growth the number of manuscripts published over the last 10 years, including applications in environmental science, food science, clinical chemistry, pharmaceutical bioanalysis, forensics, analytical biochemistry and organic synthesis. This dramatic increase of more that 100% per year can be attributed to the commercial availability of higher throughput 96-well workstations and extraction plates that allow numerous samples to be processed simultaneously. These so-called parallel-processing workstations represent the highest throughput systems currently available. The advantages and limitations of other types of systems, including discrete column systems and on-line solid-phase extraction are also discussed. Discussions of how automated solid-phase extractions can be developed, generic approaches to automated solid-phase extraction, and three noteworthy examples of automated extractions are given. The last part of the review suggests possible near- and long-term directions of automated solid-phase extraction.

Mixed-mechanism ionization to enhance sensitivity in atmospheric pressure ionization LC/MS.

A novel dual-mechanism ionization technique for LC/MS/MS has been observed, characterized and applied to the quantitation of a tertiary amine-containing drug compound in dog plasma. This mixed-mechanism ionization approach can improve the sensitivity of the pneumatically assisted electrospray experiment. Under conditions of higher than normal chromatographic flow and lower electrospray voltage, approximately a 4-fold increase in sensitivity was realized. A detection limit of 16 pg (45 fmol) on-column, and inter-day imprecision and inaccuracy of < 11 and < 15%, respectively, were obtained. A trade-off in concentration sensitivity in favor of ease of sample preparation was made to increase sample throughput. Although results strongly suggest that mixed-mechanism ionization is in operation, and that pneumatically assisted electrospray is a partial contributor to the overall ionization process, the exact nature of the second mechanism of ionization is unclear at this time.

Quantification and rapid metabolite identification in drug discovery using API time-of-flight LC/MS.

Liquid chromatography/mass spectrometry (LC/MS), utilizing a time-of-flight (TOF) mass analyzer, has been evaluated and applied to problems in bioanalysis for pharmacokinetics and drug metabolism. The data obtained by TOF MS differ from those obtained using quadrupole mass spectrometer instruments in that full-scan spectra can be routinely collected with greater sensitivity and speed. Both quantitative and qualitative information, including compound concentration in rat plasma and full-scan atmospheric pressure ionization mass spectra, are concurrently obtained. This approach has been used to characterize the disposition of several drug compounds that have been simultaneously dosed to rats in a cassette format. Quantitation limits in the 5-25 ng/mL range (approximately 20 nM) were obtained from nominal mass chromatograms (0.5 Da resolution). A reference lock mass was used to provide accurate mass measurement to reach third decimal place accuracy in the monoisotopic molecular weight. An improvement in quantitation limits was demonstrated after using accurate mass determinations. Several possible preliminary drug metabolites were confirmed or refuted, based on accurate mass. The trend of metabolite formation and clearance was qualitatively evaluated.

Semi-automated 96-well liquid-liquid extraction for quantitation of drugs in biological fluids.

A semi-automated liquid-liquid extraction (LLE) technique for biological fluid sample preparation was introduced for the quantitation of four drugs in rat plasma. All liquid transferring during the sample preparation was automated using a Tomtec Quadra 96 Model 320 liquid handling robot, which processed up to 96 samples in parallel. The samples were either in 96-deep-well plate or tube-rack format. One plate of samples can be prepared in approximately 1.5 h, and the 96-well plate is directly compatible with the autosampler of an LC/MS system. Selection of organic solvents and recoveries are discussed. Also, precision, relative error, linearity and quantitation of the semi automated LLE method are estimated for four example drugs using LC/MS/MS with a multiple reaction monitoring (MRM) approach. The applicability of this method and future directions are evaluated.

Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry.

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.

Quantitation of a novel metalloporphyrin drug in plasma by atomic absorption spectroscopy.

A bioanalytical method to quantify cobalt mesoporphyrin (CoMP), a novel therapeutic agent, in plasma has been developed and validated. The approach involves atomic absorption spectroscopy to determine total cobalt in a sample and a back-calculation of the amount of compound present. Endogenous plasma cobalt concentrations were small ( <0.2 ng/ml(-1) Co in rat plasma) in comparison to the quantitation limit (4.5 ng/ml(-1) Co). The inter-day imprecision of the method was 10.0% relative standard deviation (RSD) and the inter-day bias was +/- 8.0% relative error (RE) over a standard curve range of 4.5- 45.0 ng/ml(-1) Co. Because it quantifies total cobalt, the method cannot differentiate between parent drug and metabolites, but negligible metabolism allows reliable estimates of the actual parent drug concentration. A correlation study between the atomic absorption method and 14C-radiometry demonstrated excellent agreement (r = 0.9868, slope = 1.041 +/- 0.028, intercept = 223.7 +/- 190.0) and further substantiated the accuracy of the methods. Methodology was successfully applied to a pharmacokinetic study of CoMP in rat, with pharmacokinetic parameter estimation. The elimination half-lives, after intra-muscular and subcutaneous administration, were 7.7 and 8.8 days, respectively.

Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.

An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.

Development of a chiral HPLC method to evaluate in vivo enantiomeric inversion of an unstable, polar radiosensitizer in plasma.

A chiral HPLC method to quantify in vivo enantiomeric inversion of prodrug CI-1010 (IR) or its drug IIR (PD 146923), a radiosensitizer, upon X-irradiation of dosed rats was developed. These polar enantiomers were separated only by using normal-phase chiral HPLC. A Chiralpak AS column provided the best separation. Isolation of analytes from plasma employed solid-phase extraction (SPE), and required conditions that were compatible with normal-phase HPLC. Options for SPE were restricted by the chemically reactive nature of both prodrug and drug, which produced analyte losses as high as 100%. Acceptable recoveries using SPE required evaluation of conditions for analyte chemical stability. The validated method gave a lower-limit of quantitation (LLOQ) of 200 ng/ml for each enantiomer extracted from 0.15 ml of plasma. The LLOQ of the inverted enantiomer could be detected in the presence of 10,000 ng/ml of the dosed enantiomer. Precision (RSD) ranged from 14.2 to 4.4%, and from 24.2 to 5.1% for IIS and IIR, respectively. Accuracy (RE) was +/- 13.1 and +/- 13.2%, respectively. Recoveries ranged from 44.3 to 71.4%, and from 40.7 to 67.9%, for IIS and IIR, respectively.

Automated sample preparation for drugs in plasma using a solid-phase extraction workstation.

An automated solid-phase extraction workstation was used to develop, characterize and validate two separate HPLC methods for quantifying drugs in plasma. Method development was facilitated by workstation functions which allowed wash solvents of varying organic composition to be mixed and tested automatically. The precision estimates for the two methods were within 6.0 and 2.0% RSD across their respective calibration ranges. Accuracies for replicate determinations of quality controls were between -1.2 and +4.8 RE over ng ml-1 calibration ranges, respectively. Optimized recoveries were quantitative and were generally greater than 90% for the four analytes tested, and depended to a great extent, as expected, on the composition of the wash solvent. Sample throughput benchmarks for the two methods ranged from 3 to 10 min per sample, depending on the extent of air drying used. Because of parallel sample processing, 60 samples could be extracted in as little as 17 min.

Disposition kinetics of cobalt mesoporphyrin in mouse, rat, monkey and dog.

1. Radiometric and UV analyses indicated > 95% unchanged cobalt mesoporphyrin (CoMP) in plasma after i.v. or i.m. administration. Blood clearance of CoMP is < 2% of hepatic blood flow in mouse and rat, and < 0.5% of hepatic blood flow in monkey and dog. CoMP elimination t1/2 ranged from 3.1 to 9.9 days in animals after i.v. administration. 2. CoMP is highly (> 99.5%) bound to plasma proteins, but has low affinity for blood cells (Kp < 0.15). The volume of CoMP distribution (Vss < 0.91/kg) is reflective of a distribution to total body water following i.v. administration to mouse, rat, monkey and dog. 3. [14C]CoMP reached highest levels in rat tissue between 1 and 4 days following i.m. injection. Liver, kidney cortex, lymph node, adrenal and spleen demonstrated greatest uptake of radiolabel. Concentration in tissues was readily detectable at 60 days post-dose. 4. CoMP was slowly absorbed after i.m. administration showing dose-dependent pharmacokinetics. The major route of radiolabel elimination was faecal excretion (54% of dose) in rat after an i.m. dose of [14C]CoMP. Approximately 1% of the 14C dose was recovered in the urine over 7 days post-dose. 5. As a polar metalloporphyrin, CoMP has low clearance, restricted tissue distribution and long elimination t1/2 in the laboratory animals.

Improving ion-trap GC-MS quantitation limits for therapeutic agents extracted from rat plasma.

Insufficient quantitation limits using ion-trap gas-chromatography mass-spectrometry (GC-MS) prevented the assay of some samples during a preliminary screening of preclinical rat plasma samples (50 microliter) containing novel, polar therapeutic agents. Few options were available for improving the lower limit of quantitation. The limited amount of sample available precluded the extraction additional plasma. Lipid-liquid extraction recoveries were greater than 90% throughout the range of the standard curve (500-2000 ng ml-1). Chromatography was optimized and multiple, equivalent sites for analyte fragmentation were precluded, using MS-MS to improve assay sensitivity. Quantitation limits were decreased 10-fold however, by using a larger syringe to increase the injection volume from 5 to 50 microliter, in combination with a universal programmable injector. These large injection volumes required changes in the injector events program and in column plumbing. Additionally, evaluation of injection liner packing material demonstrated a 2-fold improvement in sensitivity, using carbofrit, relative to silanized glass wool. Converting to inert ion-trap electrodes did not appear to affect the detection limit, perhaps due to over-riding peak broadening during gas chromatography. The changes described produced a 20-fold improvement in the lower limit quantitation.

On-probe immunoaffinity extraction by matrix-assisted laser desorption/ionization mass spectrometry.

A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses.

Management of nonmatrix interfering peaks in a chiral high-performance liquid chromatographic assay produced by solid-phase extraction of rat plasma.

PD 146923, under evaluation as an alkylating radiosensitizing drug, contains one chiral center and one chemically reactive aziridine ring. A method was developed to evaluate possible in vivo enantiomeric inversion of PD 146923 in rat plasma. Normal-phase chiral HPLC was necessary to separate the enantiomers, but a typical aqueous-based solid-phase extraction (SPE) was needed to isolate the analytes from plasma. SPE at higher analyte concentrations removed all interfering peaks and gave acceptable recoveries. However, peaks (A-G) from seven new components interfering with analyte detection at lower concentrations were produced by SPE. The interfering peaks overlapped each other, so some were not observed until other, more intense interfering peaks had been managed. The low separation efficiency of the chiral column precluded management of interfering peaks by modifying chromatographic parameters. Chemical reactivity of the analytes forced the use of mild conditions for management of interfering peaks. Peaks A-F were: (A) water from the SPE cartridge; (B) SPE sorbent endcapping; (C, E and F) nonvolatile salts of the SPE elution acid reacting with bases from the injection solvent or with unidentified bases from the SPE cartridge; (D and G) analyte degradation products. This study identifies the nonmatrix peaks coeluting with the analytes, and describes how an aqueous-based SPE method was developed for isolating these very polar, highly reactive analytes in plasma for separation in a normal-phase chiral HPLC assay. Additionally, B, C, E or F probably are present in many other solid-phase extractions, but are not observed because of polarity or solubility properties.

Analytical considerations for trace determinations of drugs in breast milk.

Recent scientific and regulatory interest in lacteal excretion of drugs has prompted this review of bioanalytical sample preparation techniques for milk. The composition and properties of milk are reviewed, with emphasis on how the sample preparation is affected. The most important principals of mammary gland pharmacology, including protein binding, ion trapping and liquid solubility, are described. Because adequate milk volume is difficult to obtain from some smaller rodent species, special arrangements for sample collection, control preparation and assay standardization often need to be made. Several commonly-used sample preparation approaches for drugs in milk, including direct injection, dialysis and ultrafiltration, protein precipitation, liquid-liquid extraction, solid-phase extraction and immunoaffinity extraction. Have been reported with varying degrees of success. The advantages and disadvantages of each of these approaches is discussed.

Tandem-in-time mass spectrometry as a quantitative bioanalytical tool.

Tandem-in-time mass spectrometry, as implemented on an ion-trap detector (ITD), is the process whereby precursor ions are created, stored in a radio frequency (rf) trapping field, and then sequentially fragmented to form product ions by application of additional rf waveforms. As with any form of tandem mass spectrometry (MS/MS), tandem-in-time MS is highly selective, by virtue of both mass discrimination and specific gas-phase chemistry. Beyond this, however, tandem-in-time MS offers ion throughput efficiency and cost advantages over either quadrupole or sector instruments. This paper will describe the use of capillary gas chromatography combined with tandem-in-time mass spectrometry to quantify a novel therapeutic agent extracted from human plasma. For an example compound, a quantitation limit of 25 pg/mL (S/N approximately 10, 15 fmol on-column) was attained out of plasma. The interday imprecision was < or = 12.2% over a dynamic range extending to 10 ng/mL. Due to favorable ionization conditions for the test analytes, electron ionization resulted in formation of M+ ions, with very little fragmentation, allowing for maximum assay sensitivity. Although method characterization and validation demonstrated adequate instrumental performance, some lack of ruggedness was encountered during routine application.