Biomarker Assay Validation by Mass Spectrometry.

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.

Unconjugated payload quantification and DAR characterization of antibody-drug conjugates using high-resolution MS.

AIM: The application of high-resolution MS to antibody-drug conjugate (ADC) drug development may provide insight into their safety and efficacy. Quantification of unconjugated cytotoxic drug (payload) and characterization of drug-to-antibody ratio distribution were determined in plasma using orthogonal acceleration quadrupole-time-of-flight MS. RESULTS: Unconjugated payload quantification determined by quadrupole-time-of-flight-based MRM(highresolution) and triple quadrupole-based multiple reaction monitoring were comparable and achieved detection limits of 0.030 and 0.015 ng/ml, respectively. As determined by immunocapture and TOF-MS, drug-to-antibody ratio remained unchanged up to 3-weeks postdose for an ADC containing engineered glutamine linkers, but declined from four to three over 2 weeks in an ADC containing engineered cysteine linkers. CONCLUSION: The use of high-resolution MS in ADC drug discovery confirms its utility within the bioanalytical discipline.

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.

Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry.

We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.

Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC-MS/MS.

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC-MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C(18) column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.

Polymeric nanoparticles for increased oral bioavailability and rapid absorption using celecoxib as a model of a low-solubility, high-permeability drug.

PURPOSE: To demonstrate drug/polymer nanoparticles can increase the rate and extent of oral absorption of a low-solubility, high-permeability drug. METHODS: Amorphous drug/polymer nanoparticles containing celecoxib were prepared using ethyl cellulose and either sodium caseinate or bile salt. Nanoparticles were characterized using dynamic light scattering, transmission and scanning electron microscopy, and differential scanning calorimetry. Drug release and resuspension studies were performed using high-performance liquid chromatography. Pharmacokinetic studies were performed in dogs and humans. RESULTS: A physical model is presented describing the nanoparticle state of matter and release performance. Nanoparticles dosed orally in aqueous suspensions provided higher systemic exposure and faster attainment of peak plasma concentrations than commercial capsules, with median time to maximum drug concentration (Tmax) of 0.75 h in humans for nanoparticles vs. 3 h for commercial capsules. Nanoparticles released celecoxib rapidly and provided higher dissolved-drug concentrations than micronized crystalline drug. Nanoparticle suspensions are stable for several days and can be spray-dried to form dry powders that resuspend in water. CONCLUSIONS: Drug/polymer nanoparticles are well suited for providing rapid oral absorption and increased bioavailability of BCS Class II drugs.

Simultaneous and high-throughput quantitation of urinary tetranor PGDM and tetranor PGEM by online SPE-LC-MS/MS as inflammatory biomarkers.

Quantitation of urinary tetranor PGDM or tetranor PGEM (tPGDM and tPGEM) in the past was performed separately using off-line SPE LC-MS/MS methods. The manual SPE procedure is generally time-consuming and cost-ineffective. In addition, simultaneous quantitation of tPGDM and tPGEM is favorable yet very challenging because of the similar chemical structures and identical MRM transitions. This work describes the development and validation of a high-throughput online SPE-LC-MS/MS method, allowing simultaneous and high-throughput measurement of tPGDM and tPGEM in human urine. The reportable range of the assay was 0.2-40 ng/ml for tPGDM and 0.5-100 ng/ml for tPGEM. Intra- and inter-assay precision and accuracy determined using quality control samples were all within acceptable ranges (% CV and % Bias < 15%). Tetranor PGDM was stable under all tested conditions while tPGEM was stable at 4 °C and after three F/T cycles but not stable at room temperature for 24 h (recovery below 80%). The assay was applied to measure urinary tPGDM and tPGEM among healthy volunteers, smokers and COPD patients. Significantly higher urinary levels of both tPGDM and tPGEM were observed in COPD patients than those of non-smoking healthy volunteers. These results demonstrated that the high-throughput online SPE-LC-MS/MS assay provides sensitive, reproducible and accurate measurement of urinary tPGDM and tPGEM as biomarkers for assessing inflammatory diseases such as COPD.

Development and validation of a sample stabilization strategy and a UPLC-MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF).

A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.

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%.

Automated sample preparation for regulated bioanalysis: an integrated multiple assay extraction platform using robotic liquid handling.

BACKGROUND: A novel approach for regulated bioanalytical sample preparation has been developed to combine multiple types of extraction techniques into one integrated and automated sample-preparation suite that pairs a graphical user interface with the Hamilton Microlab(®) STAR robotic liquid handler. RESULTS: The multi-assay sample-preparation suite is composed of three bioanalytical extraction techniques: protein precipitation, solid-phase extraction and liquid-liquid extraction. Validation data provided highly reproducible and robust results for each respective automated extraction technique. CONCLUSION: The user-friendly graphical user interface and modular method design provide a flexible and versatile approach for routine bioanalytical sample-preparation and is the first fully integrated multiple assay sample-preparation suite for regulated bioanalysis.

Quantification of urinary PGEm, 6-keto PGF(1alpha) and 2,3-dinor-6-keto PGF(1alpha) by UFLC-MS/MS before and after exercise.

Eicosanoids play an important role in the evaluation of pro-inflammatory responses and in the safety and toxicity of novel therapeutic agents. This work describes a high-throughput UFLCMS/MS method for the analysis of three urinary prostanoid biomarkers of pro-inflammatory responses, tetranor PGEm, 6-keto PGF(1alpha) and 2,3-dinor-6-keto PFG(1alpha). Nine male volunteers of various age and fitness level participated in this study. Six provided pre- and post-exercise samples and three provided intraday samples. Tetranor PGEm and 6-keto PGF(1alpha) increased significantly in patients after exercise (p<0.017 and p<0.029). In individual patient sets, tetranor PGEm levels increased from 1.5- to 6-fold pre- vs. post-exercise, levels of 6-keto PGF(1alpha) increased more dramatically from 2- to 55-fold pre- vs. post-exercise. The prostanoid 2,3-dinor-6-keto PGF(1alpha) remained unchanged post-exercise. Data was normalized to urinary creatinine concentration, which increased approximately 40% post-exercise.

Detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for biofluid analysis and tissue imaging.

Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO(3) on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desorption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na(+) cationization of carbohydrates and Ag(+) cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1-200 microM). Additionally, we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.

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.

Nanostructure initiator mass spectrometry: tissue imaging and direct biofluid analysis.

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1'-hydroxylation kinetics in human liver microsomes.

The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.

Mechanism-based inactivation of cytochrome P450 2D6 by 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]- 4-[4-(trifluoromethyl)-2-pyridinyl]piperazine: kinetic characterization and evidence for apoprotein adduction.

The kinetics for inactivation of cytochrome P450 2D6 by (1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) were characterized, and the mechanism was determined in an effort to understand the observed time-based inactivation. Loss of dextromethorphan O-demethylase activity following coincubation with EMTPP followed pseudo-first-order kinetics and was both NADPH- and EMTPP-dependent. Inactivation was characterized by an apparent Ki of 5.5 microM with a maximal rate constant for inactivation (kinact) of 0.09 min(-1), a t1/2 of 7.7 min, and a partition ratio of approximately 99. P450 2D6 inactivation was unaffected by coincubation with exogenous nucleophiles or reactive oxygen scavengers and was protected by the competing inhibitors N-4-(trifluoromethyl)benzyl quinidinium bromide and quinidine. After a 30 min incubation with 100 microM EMTPP, dextromethorphan O-demethylase activity was decreased approximately 76%, with a disproportionate loss ( approximately 35%) in carbon monoxide binding. Additional mechanistic studies showed no evidence of either metabolite inhibitory complex formation or heme adduction. However, a P450 2D6 apoprotein adduct was characterized that had a mass shift relative to unadducted P450 2D6 apoprotein consistent with the molecular mass of EMTPP (353 Da). In vitro metabolism studies revealed that EMTPP is susceptible to P450 2D6-mediated hydroxylation and dehydrogenation, postulated to both form via initial hydrogen atom abstraction from the alpha-carbon of the imidazole ethyl substituent. Additional studies demonstrated that while a dehydrogenated EMTPP metabolite was apparently stable and observable, we propose that a thermodynamic partitioning may exist, which results in formation of a second dehydrogenated imidazo-methide-like metabolite that may serve as the reactive species causing mechanism-based inactivation of P450 2D6. Last, trapping studies with EMTPP yielded an N-acetyl cysteine conjugate, which upon tandem MS and NMR analysis revealed adduction to the alpha-carbon of the imidazole ethyl substituent. Overall, evidence suggests that nucleophilic attack of an imidazo-methide-like intermediate by a P450 2D6 active site residue leads to apoprotein adduction and consequent inactivation.