Quantification of fumarate and investigation of endogenous and exogenous fumarate stability in rat plasma by LC-MS/MS.

BACKGROUND: Fumaric acid is a commonly used excipient in pharmaceutical products. It is not known if its presence may lead to fluctuation of endogenous fumarate levels. An LC-MS/MS method was developed and validated to quantify fumarate in support of a toxicokinetics study. RESULTS: Stability evaluation showed that endogenous fumarate was stable for 6 h at room temperature, while exogenously added fumaric acid was converted to malate within 1 h due to the presence of fumarase. Citric acid, a fumarase inhibitor, prevented the conversion of added fumaric acid in rat plasma. CONCLUSION: The method was validated in citric acid stabilized rat plasma using a surrogate matrix approach. A discrepancy in stability was observed between endogenous fumarate and exogenously added fumaric acid.

Small molecule specific run acceptance, specific assay operation, and chromatographic run quality assessment: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization teams.

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

Accurate weighing and dilution-assisted plasma microsampling (AWADA-PM): an easy-to-implement and rugged strategy.

BACKGROUND: Liquid microsampling can realize ethical benefits through reduced animal usage. It inherently deals with a minute amount of sample; for example, <30 µl of plasma, which is generally insufficient for multiple analyses. RESULTS: We report accurate weighing and dilution-assisted plasma microsampling (AWADA-PM) that substantially increases sample sizes (e.g., by tenfold). Plasma samples are harvested from blood samples (~70 µl) in capillaries. The plasma samples are weighed with a calibrated balance. The weights are converted to volumes using a standard plasma density of 1.025 g/cm(3). Diluent is added with a calibrated pipette to the harvested plasma to achieve a tenfold dilution. CONCLUSION: The proof-of-concept for AWADA-PM was successfully demonstrated for the quantitation of acetaminophen in rat plasma (K2EDTA) using liquid chromatography-tandem mass spectrometry employing water and blank rat plasma as diluents. The results clearly demonstrate that the AWADA-PM strategy is an easy-to-implement and reliable microsampling technology that has potential for full regulatory compliance.

Systematic evaluation of supported liquid extraction in reducing matrix effect and improving extraction efficiency in LC-MS/MS based bioanalysis for 10 model pharmaceutical compounds.

In past a few years, there has been a large increase in the application of supported liquid extraction (SLE) for LC-MS/MS based bioanalysis due to its distinct practical advantage in reduced time cost, ease of operation and the feasibility for automation. The main purpose of this study was to systematically evaluate supported liquid extraction in reducing matrix effect and improving extraction efficiency/recovery under various extraction conditions with 10 model pharmaceutical compounds in liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) analysis. Selected compounds have diverse physicochemical properties where logP ranges from 0.1 to 6.24 and pK(a) ranges from 4.0 to 11.1. The factors that may have the impact on the recovery of analytes and phospholipids (PL) were assessed. Over 75% recovery was achieved for every analyte under its respectively optimized extraction conditions where the selection of the polarity of extraction solvent and buffered pH can be critical for efficient recovery. Furthermore, the matrix effect was assessed by postextraction spike and postcolumn infusion method. The matrix effect was considerably reduced for all analytes under most extraction conditions evaluated for SLE, compared with protein precipitation (PPT) method. The correlation between matrix effect and residual phospholipids in sample extract was clearly shown. Although analyte-dependent matrix effect was observed prominently in sample extract prepared by PPT, it was minimized by SLE sample preparation process that effectively removes the majority of phospholipids. Sample extracted by ethyl acetate contained more phospholipids and demonstrated stronger matrix effect than by other organic solvents. Water-miscible organic content, such as methanol and acetonitrile in samples prior to loading has significant impact on PL recovery when eluting with methyl tert-butyl ether. However, isopropanol does not enhance the recovery of PL when adding to dichloromethane for elution. In addition, the compromise between improved extraction efficiency by SLE and reduced matrix effect is sometimes necessary to yield clean extract with acceptable recovery. The effective removal of phospholipids and reduction of matrix effect, while achieving good recovery for all pharmaceutical compounds with diverse physicochemical properties, demonstrated that SLE is a valuable alternative technique to liquid-liquid extraction (LLE) in high throughput LC-MS/MS based bioanalysis.

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma.

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6ß-naloxol (NLL) and naloxone-3ß-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 µm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.

Determination of carboplatin in human plasma using HybridSPE-precipitation along with liquid chromatography-tandem mass spectrometry.

The main purpose of this study was to develop and validate a rapid, specific, sensitive, and reliable LC-MS/MS-based bioanalytical method for the determination of carboplatin in human plasma. The optimal chromatographic behavior of carboplatin was achieved on a Biobasic SCX column (50 mm × 2.1 mm, 5 µm) using ion exchange chromatography. The total LC analysis time per injection was 2.6 min with a flow rate of 1.5 mL/min with a gradient elution. Optimization with regard to improving recovery and minimizing matrix effects using HybridSPE-precipitation (HybridSPE-PPT) has been evaluated under various extraction conditions. As a result, sample preparation via HybridSPE-PPT with 1% formic acid in acetonitrile in a 96-well format was applied for method validation and sample analysis and showed acceptable recovery of greater than 25% and negligible matrix effects. The method validation was conducted over the curve range of 2.00-2000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.8% relative standard deviation (RSD) and -13.2 to -3.6% relative errors (RE). The method was successfully applied to determine carboplatin in human plasma samples.

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay.

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC-MS/MS (2D-LC-MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00-250 pg/ml for human plasma and 50.0-10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.

2009 White Paper on recent issues in regulated bioanalysis from the 3rd Calibration and Validation Group Workshop.

The 3rd Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis was organized by the Calibration and Validation Group as a 1.5-day full immersion workshop for contract research organizations, pharmaceutical companies and regulatory agencies to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges. A consensus was reached among panelists and attendees on many points regarding method validation of small molecules.

Comparison of fused-core and conventional particle size columns by LC-MS/MS and UV: application to pharmacokinetic study.

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC-MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C(18) column (5 microm, 2.0 mm x 30 mm) and a fused-core C(18) column (2.7 microm, 2.1 mm x 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2-3-fold increased efficiency (N). Loading capacity up to 25 microl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2-5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 microm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC-MS/MS. The calibration curves were linear in the concentration range of 0.5-1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within +/-7.8% and +/-7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.

Workshop report and follow-up--AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples--implications of Crystal City recommendations.

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR. These recommendations from the Workshop, along with the subsequent evolution of approaches leading to a robust ISR program, may be used by scientists performing bioanalytical assays for regulated studies to provide additional confirmation of assay reproducibility for incurred samples.

Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma.

A highly selective, sensitive and robust LC-MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M+2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 microm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within +/-15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.

Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma.

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine.

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry.

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.