Christopher A. James

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS

INTRODUCTION Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia. METHODS An LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays. RESULTS The calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at -60 to -80 degrees C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were -11.2% and 5.6%, respectively at the LLOQ, and less than +/-7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between -0.7 and +2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n=16, median 121 ng/mL) and 99 ng/mL (n=50, median 68 ng/mL), respectively. CONCLUSIONS A fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples.

Simultaneous Analysis of Multiple Monoclonal Antibody Biotherapeutics by LC-MS/MS Method in Rat Plasma Following Cassette-Dosing

We have recently developed a general liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using a stable isotope-labeled (SIL) monoclonal antibody (mAb) as an internal standard (IS) for single-analyte quantification of mAb (Li et al. Anal Chem 84(3):1267–1273, 2012). The method offers an advantage over ligand binding assay in reducing the time and resources needed for bioanalytical support in preclinical stages of drug development. In this paper, we report another marked increase in assay efficiency for multi-analyte bioanalysis using unique surrogate peptides for each analyte and the strategic choice of the SIL-IS peptide. The method was qualified for the simultaneous determinations of four mAbs in rat plasma and applied to samples from discrete- and cassette-dosed rats. The pharmacokinetic parameters of the four mAbs of cassette dosing were comparable to those of discrete dosing and of enzyme-linked immunosorbent assay results. Although there may be limitations and special considerations for cassette-dosing of biologics, these results demonstrate the robust performance of the multi-analyte LC-MS/MS method allowing cassette-dosing that would ultimately reduce animal use and improve efficiency.

Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC-MS/MS.

An LC-MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50-10,000 ng/mL and ¹³C₂¹⁵N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine. Interday bias (% RE) and precision (% CV) were -4.2 and 12.3% at the LLOQ, and less than ±0.9 and 8.3% for higher concentrations, respectively. Glycine was stable in artificial CSF for at least 5h at room temperature, 55 days at -70 °C (-60 to -80 °C range), and through three freeze-thaw cycles.

Application of automated dried blood spot sampling and LC-MS/MS for pharmacokinetic studies of AMG 517 in rats

BACKGROUND The use of dried blood spot (DBS) sampling technique is of particular interest for drug discovery pharmacokinetic studies due to the small blood volume requirement. In addition, automated blood sampling is an attractive approach for rat pharmacokinetic studies as animal handling work is minimized. The goal of this study was to use an automated DBS sampler for automated blood collection and spotting onto DBS paper for pharmacokinetic studies in rats. AMG 517, a potent and selective vanilloid receptor antagonist, was dosed to rats (n = 3) intravenously and blood samples were collected at nine time points over a 24 h period using the automated DBS sampler. After drying, storage and shipment, the DBS samples were extracted and analyzed by LC-MS/MS. RESULTS The developed bioanalytical method for the analysis of DBS samples had good accuracy and precision within the context of a discovery, non-GLP analysis. The concentration-time data and pharmacokinetic parameters generated from automated spotted samples were very similar to those derived from manually spotted DBS samples. The manual DBS data were also comparable to plasma data after correction for blood-to-plasma ratio. CONCLUSION The automated DBS sampling is a promising technique for rodent pharmacokinetic studies and will improve the efficiency and quality of DBS sampling.

Procedural elements involved in maintaining bioanalytical data integrity for good laboratory practices studies and regulated clinical studies.

This article describes procedural elements involved in ensuring the integrity of bioanalytical data. These elements can be divided into 3 areas. First, there are those ensuring the integrity of the analyte until analysis, through correct sample collection, handling, shipment, and storage procedures. Incorrect procedures can lead to loss of analyte via instability, addition of analyte through contamination or instability of related metabolites, or changes in the matrix composition that may adversely affect the performance of the analytical method. Second, the integrity of the sample identity needs to be maintained to ensure that the final result reported relates to the individual sample that was taken. Possible sources of error include sample mixup or mislabeling, or errors in data handling. Finally, there is the overall integrity of the documentation that supports the analysis, and any prestudy validation of the method. This includes a wide range of information, from paper and electronic raw data, through standard operating procedures and analytical procedures and facility records, to study plans and final reports. These are critical to allow an auditor or regulatory body to reconstruct the study.

Implementing Dried Blood Spot Sampling for Clinical Pharmacokinetic Determinations: Considerations from the IQ Consortium Microsampling Working Group

This paper was developed with the support of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). IQ is a not-for-profit organization of pharmaceutical and biotechnology companies with a mission of advancing science-based and scientifically driven standards and regulations for pharmaceutical and biotechnology products worldwide. Within the IQ, various working groups (WG) have been formed, where the microsampling WG is committed to providing a scientific forum for the advancement of both wet and dry microsampling techniques within the pharmaceutical industry. This first output from the microsampling WG is to summarize and reflect on the current knowledge and opinions on DBS sampling, to stimulate discussion, and to encourage future creative applications of DBS sampling. Dried blood spot (DBS) sampling has established itself as an innovative sampling technique where wet blood is spotted onto absorbent paper or other paper materials and allowed to dry (1–4). DBS offers several potential benefits inherent to the technique, namely a low blood volume, simplified blood sample collection (5), and convenient sample storage and transfer. In certain applications, DBS sampling has been shown to stabilize certain analytes or metabolites without the addition of chemical modifiers (6–9). DBS has been routinely applied for decades in neonatal screening for phenylketonuria and other congenital metabolic disorders (10). The utility of DBS sampling has also been demonstrated for therapeutic drug monitoring (11) and for epidemiological studies (e.g., HIV and HBV detection/monitoring) (12) due to the practical advantages along with simplified sample collection and handling procedures. Finally, DBS can also be used for quantitative biomarker (PD) assessment from blood, where appropriate. However, the technique is relatively new to the pharmaceutical industry and to government regulators overseeing new drug applications. Nevertheless, over the past 5 to 7 years, the technique has been extensively evaluated for quantifying drug exposure in nonclinical and/or clinical studies in various stages of drug discovery and development. The ease to collect, transfer, store, and process small volumes of blood samples has generated considerable interest in providing utility in volume-limited situations (e.g., small rodent, human pediatric studies) for toxicokinetic (TK), pharmacokinetic (PK), or pharmacodynamic (PD) sampling. Discovery and nonclinical studies Rodent animal models are typically employed in these studies. The reduced blood volumes required for DBS can enable serial bleeding and, consequently, elimination of satellite animal groups and reduction of compound use. The ability to eliminate the satellite animal groups enables the assessment of exposure and toxic effects within the same animal. Studies involving expensive animal models (i.e., transgenic mice, knock-out mice, humanized mice, etc.) further highlight a persuasive scientific and economic case for DBS sampling since a complete pharmacokinetic profile can be obtained from a single study animal without the need for extra rodents merely for generating exposure data. These are perfectly in line with the principles of the 3Rs: reduction, refinement, and replacement of humane animal research (13–15). With greater emphasis from the regulatory authorities to study new drugs for infants, neonates, and pediatric populations, the requirement to conduct associated nonclinical juvenile rodent toxicity studies serves as an ideal scenario where the advantage of low blood volume in DBS sampling is undeniable. Although the advantages of DBS heavily favor rodent studies, it can also be used to refine non-rodent studies.

Direct Quantitative Analysis of a 20 kDa PEGylated Human Calcitonin Gene Peptide Antagonist in Cynomolgus Monkey Serum Using In-Source CID and UPLC-MS/MS

PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented. CGRP[Cit, Cit] is an investigational human calcitonin gene peptide receptor antagonist with amino acid sequence Ac-WVTH[Cit]LAGLLS[Cit]SGGVVRKNFVPT DVGPFAF-NH2. In-source collision-induced dissociation (in-source CID) of 20 kDa PEGylated peptide was used to generate CGRP[Cit, Cit] fragment ions, among which the most abundant b8+ ion was selected and measured as a surrogate for the 20 kDa PEGylated peptide. A solid phase extraction (SPE) method was used to extract the PEGylated peptides from the biomatrix prior to the UPLC-MS/MS analysis. This method achieved a lower limit of quantitation (LLOQ) of 5.00 ng/mL with a serum sample volume of 100 μL, and was linear over the calibration range of 5.00 to 500 ng/mL in cynomolgus monkey serum. Intraday and interday accuracy and precision from QC samples were within ±15%. This method was successfully applied to a pharmacokinetic study of the 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkeys.

Development and validation of a method for the determination of a therapeutic peptide with affinity for the human B1 receptor in human plasma using HPLC-MS/MS

The peptide described in this report (MW 1180 Da; 10-amino acid synthetic peptide) is a potent and selective antagonist of the human B1 receptor (B1) that has been investigated for the treatment of chronic pain. A method to quantitate this peptide in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics, and efficacy of this compound. Plasma samples (0.2 mL) were extracted using a Waters Oasis MAX (10 mg) 96-well plate and the resulting samples were analyzed using an Applied Biosystems API-5000 HPLC-MS/MS with an electrospray ionization (ESI) source. The method was validated for the determination of the B1 peptide in human plasma over the concentration range of 1-50 ng/mL. Isotopically labeled B1 peptide ((13)C6(15)N(2)-B1 peptide) was used as an internal standard. Interday precision and accuracy, determined from analysis of quality control (QC) samples, yielded coefficients of variation (CV) of less than 5.3% and accuracy within a 2.4%. Within batch precision and accuracy determinations provided CV values of less than 7.3% and accuracy within a 6.0% bias. Precautions had to be taken to prevent B1 peptide loss to container surfaces and contamination of the HPLC-MS/MS. The validated assay was used in support of human clinical trials.

Application of DBS sampling in combination with LC–MS/MS for pharmacokinetic evaluation of a compound with species-specific blood-to-plasma partitioning

BACKGROUND Dried blood spot (DBS) sampling in combination with LC-MS/MS has been used increasingly in drug discovery for quantitative analysis to support pharmacokinetic (PK) studies. In this study, we assessed the effect of blood-to-plasma (B:P) partitioning on the bioanalytical performance and PK data acquired by DBS for a compound AMG-1 with species and concentration-dependent B:P ratio. RESULTS B:P partitioning did not adversely affect bioanalytical performance of DBS for AMG-1. For rat, (B:P ratio of 0.63), PK profiles from DBS and plasma methods were comparable. For dog, concentration-dependence of B:P ratio was observed both in vivo and in vitro. Additional studies demonstrated concentration-dependence of the compounds unbound fraction in plasma, which may contribute to the concentration-dependence of the B:P ratio. CONCLUSION DBS is a promising sampling technique for preclinical pharmacokinetic studies. For compounds with high B:P ratio, caution needs to be applied for data comparison and interpretation between matrices.

Simultaneous determination of a p38 MAP kinase inhibitor and its amide hydrolyzed metabolite in Cynomolgus monkey plasma by LC-MS/MS, and its application to a toxicokinetic study.

A LC-MS/MS method was developed for the determination of a p38 MAP kinase inhibitor (Compound I) and its amide hydrolyzed metabolite (M7) in Cynomolgus monkey plasma over the concentration range of 1.00-1000ng/mL. Stable isotope labeled compounds (d(3)-Compound I and d(3)-M7) were used as internal standards (IS). Samples were prepared using protein precipitation in the 96-well format with a 30μL plasma sample volume. Chromatographic separation was performed with reversed-phase liquid chromatography on a Varian Monochrom C(18) (100mm×2.00mm, 5μm) analytical column. The mobile phases were 5mM ammonium formate in acetonitrile/water (95/5, v/v) pH 7.0 and 5mM ammonium formate in acetonitrile/water (5/95, v/v) pH 7.0. Gradient elution, at a flow rate of 550μL/min, was used to separate Compound I and M7. Positive atmospheric pressure chemical ionization was utilized with detection by multiple reaction monitoring (MRM). Total run time was about 3.2min. This method was validated following the current Food and Drug Administration (FDA) guidance for bioanalytical method validation. The intra- and inter-day precision (% CV) and accuracy (% bias) at all concentrations tested were below 15% for both analytes. The mean recoveries for Compound I, M7, d(3)-Compound I, and d(3)-M7 were 106%, 107%, 108% and 105%, respectively. The method was successfully applied to support a GLP toxicokinetic study in Cynomolgus monkeys after oral administration of Compound I. A total of 48 samples (∼12.5% of the total number of samples) were selected for incurred sample reanalysis (ISR). The % difference between the reassay concentrations and the original concentrations were all less than 20% of their mean values and met the acceptance criteria for ISR.