Chester L. Bowen

Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry

A novel approach to the quantitative determination of xenobiotics in whole blood samples without sample preparation or chromatography is described. This method is based on direct analysis of microlitre volumes of blood which are spotted onto specialized paper cards and dried, with the resulting dried blood spots (DBS) analyzed directly via desorption electrospray ionization (DESI) mass spectrometry (MS). Using sitamaquine, terfenadine, and prazosin as model compounds with verapamil as a common internal standard, this methodology demonstrated detection of each compound down to 10 ng mL(-1) from DBS where standard calibration curves show linearity from 10-10,000 ng mL(-1) with r(2) > 0.99. Three (3) different untreated types of filter papers (Whatman 903 and 31ETF as well as Ahlstrom 237) and two (2) treated types of filter paper (Whatman FTA and FTA Elute) were examined and the effect of each surface on the recovery of each analyte was evaluated. The results show that the untreated papers provide the best substrates for DBS analysis by DESI. A more in depth study of the quantitation of sitamaquine on 31ETF paper stock provided bias and error measurements of less than 20%. The promising results shown in this study may have important implications in the areas of therapeutic drug monitoring (TDM), clinical and forensic toxicology, and pharmacology.

Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds

BACKGROUND Compound stability remains a major point of concern within pharmaceutical development. In attempts to minimize degradation, scientists may utilize acidification of samples prior to storage, dark chambers, decreased freezer temperatures and a variety of other stabilization techniques. All of these steps require additional procedures, increased costs and increased validation steps. Dried blood spots (DBS) are becoming a popular alternative to plasma sampling in many small- and even large-molecule applications. An investigation was performed in order to establish if DBS would provide storage advantages over liquid-based matrices for two light-sensitive compounds, nifedipine and omeprazole, to prevent or minimize photodegradation. RESULTS Experimental data has shown, through forced and natural photodegradation experiments, that the compounds nifedipine and omeprazole exhibit increased photostability when spotted and stored on various DBS paper, when compared with water, plasma or whole blood. For omeprazole, between 40 and 90% loss was observed in liquid matrices, while photodegradation was negligible when utilizing DBS. Some loss of nifedipine is noted during exposure conditions on DBS; however, photodegradation in liquid matrices is far more severe. CONCLUSION Within the experimental compound set, DBS technology offers a significant reduction in the photodegradation process when compared with the liquid matrices water, plasma or blood.

Development of a sensitive and selective LC-MS/MS method for simultaneous determination of gemcitabine and 2,2-difluoro-2-deoxyuridine in human plasma.

A sensitive, selective, and quantitative method for the simultaneous determination of gemcitabine and 2,2-difluoro-2-deoxyuridine (dFdU) has been developed and validated in human plasma in the presence of tetrahydrouridine, a cytidine deaminase inhibitor. The method employs derivatization of gemcitabine and dFdU with dansyl chloride to improve the chromatographic retention and separation. The derivatization was performed in plasma without prior sample clean-up, followed by extraction of the dansyl-derivatives using methyl tertiary-butyl ether (MTBE). Ultra performance liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 microm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration ranges of 20-5000 and 100-25,000 ng/mL for gemcitabine and dFdU, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support pharmacokinetic studies. In addition, the relatively small sample volume (50 microL) and a run time of 1.5 min facilitate automation and allow for high-throughput analysis.

Application of DBS for quantitative assessment of the peptide Exendin-4; comparison of plasma and DBS method by UHPLC–MS/MS

BACKGROUND An investigation was performed in order to establish if dried blood spots (DBS) could be applied to the quantitation of biopharmaceuticals in biological matrices and perform equivalently in terms of accuracy, precision and stability to traditional plasma methods. RESULTS A method was successfully validated for the peptide Exendin-4 (39 amino acids in length) utilizing DBS technology. The validated DBS method resulted in a more sensitive and simplistic method than an existing monkey plasma method and required tenfold less sample volume. The final DBS method resulted in a 10-2000-ng/ml linear calibration range using approximately 5 µl of dried blood, compared with the plasma method in which 150 µl of plasma coupled with SPE sample preparation resulted in a 20-2000-ng/ml linear calibration range. Although not needed for DBS, SPE was required for the plasma method to reduce endogenous matrix interferences and achieve desired LLOQ. Matrix stability was also enhanced by the implementation of the DBS platform when compared with either plasma or whole blood. CONCLUSION DBS technology can be utilized for the quantitation of biopharmaceuticals and offer advantages over traditional plasma-based methods.

A novel approach to capillary plasma microsampling for quantitative bioanalysis

BACKGROUND A novel device and procedure for the collection and isolation of microvolumes of plasma have been developed and two pilot rodent PK studies have been completed. RESULTS This method involves collection of blood into a plastic-wrapped, EDTA-coated capillary tube, containing a small amount of a thixotropic gel and a porous plug. Following blood collection, the capillary is placed into a secondary labeled container suitable for centrifugation and plasma is generated. During centrifugation, the thixotropic gel isolates the plasma from the red blood cells and creates a physical barrier between the two matrices. The plasma is then dispensed from the capillary tube into a separate container for storage or processing. CONCLUSION A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.

Development and validation of a sensitive and selective UHPLC–MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.

The metabolic drug-drug interaction profile of Dabrafenib: in vitro investigations and quantitative extrapolation of the P450-mediated DDI risk.

Dabrafenib is a potent ATP-competitive inhibitor for the V600 mutant b-rapidly accelerated fibrosarcoma (b-raf) kinase currently approved in the United States for the treatment of metastatic melanoma. Studies were conducted in human liver microsomes, recombinant human cytochrome P450 (P450) enzymes, and human hepatocytes to investigate the potential of dabrafenib and its major circulating metabolites to perpetrate pharmacokinetic drug-drug interactions (DDIs) as well as have their own pharmacokinetics affected (victim) by coadministered drugs. Dabrafenib metabolism was mediated by CYP2C8 (56% to 67%) and CYP3A4 (24%); in addition, it has demonstrated inhibition of CYP2C8, 2C9, 2C19, 3A4 (atorvastatin), and (nifedipine), with calculated IC50 values of 8.2, 7.2, 22.4, 16, and 32 μM. It also demonstrated metabolism-dependent inhibition of CYP3A4 with a maximal inactivation rate constant of 0.040 minute−1 and a concentration required to achieve half-maximal inactivation for CYP3A4 of 38 μM. Hydroxy-dabrafenib inhibited CYP1A2, 2C9, and 3A4 (midazolam) with calculated IC50 values of 83, 29, and 44 μM, and carboxy-dabrafenib did not inhibit any of the P450 enzymes tested. Desmethyl-dabrafenib inhibited CYP2B6, 2C8, 2C9, 2C19, and 3A4 (midazolam, atorvastatin, and nifedipine) with calculated IC50 values of 78, 47, 6.3, 36, 17, 20, and 28 μM, respectively. At 30 μM dabrafenib showed increases in CYP2B6 and CYP3A4 mRNA expression indicative of induction. The potential clinical relevance of these findings was explored by using mechanistic static mathematical models to estimate the magnitude of change (area under the curve change) as a result of P450-mediated DDI interactions. This risk-assessment approach indicated that dabrafenib is unlikely to perpetrate any in vivo DDIs by inhibition mechanisms, but is a likely inducer of CYP3A4 and a victim of CYP3A4 and CYP2C8 inhibitors. Furthermore, inclusion of the in vitro drug interaction data for dabrafenib metabolites did not impact the overall clinical risk assessment.

Evaluation of glucuronide metabolite stability in dried blood spots

BACKGROUND Stabilization of phase II metabolites is an important consideration during bioanalytical method development, method validation and sample analysis. Generic approaches to stabilize these metabolites during storage in liquid-based matrices include pH adjustment of samples prior to storage and/or temperature control; although a variety of other compound-specific stabilization techniques exist. Dried blood spot (DBS) technology is becoming a popular alternative to liquid matrix sampling in many preclinical and clinical applications. However, concerns remain regarding the stability of metabolites stored under ambient conditions using DBS. RESULTS Experimental data have shown that, under ambient storage conditions, the stability of the glucuronides investigated herein stored as DBS is equivalent to that of liquid samples stored at -80°C. CONCLUSION The decision to employ DBS technology for a given study needs to be considered on a case-by-case basis with an understanding of compound-specific metabolism characteristics and clinical study design.

Investigations into the environmental conditions experienced during ambient sample transport: impact to dried blood spot sample shipments

BACKGROUND Prior to bioanalysis, sample transport and storage are critical considerations in any pharmacokinetic or toxicokinetic study design. Care must be taken to ensure the shipment is properly packaged and tracked to make certain it arrives at the desired, final destination in the appropriate timeframe, and that the integrity of the sample is not compromised. When dealing with biological specimens, environmental conditions may have a deleterious effect on the stability and conditions of the sample. RESULTS Currently, frozen plasma or blood samples are the matrix of choice within the pharmaceutical industry for analysis within both preclinical and clinical trials. Liquid samples are shipped and received frozen and, therefore, the assumption is made that the frozen conditions are maintained throughout the entire transit process. Dried blood spot and dried matrix spot samples are becoming popular alternatives to plasma sampling in many small- and even large-molecule applications. With the implementation of dried blood spot and dried matrix spot samples, shipping and storage occurs under ambient conditions. CONCLUSION In this article we discuss various shipping containers for these samples, illustrate the environmental extremes encountered during the shipping process, demonstrate a cost-effective method of monitoring both temperature and humidity, and discuss validation steps that may be implemented to minimize the impact of these variables on your study design.

Modify on the fly: triple quad to high resolution in support of a dermal clinical study requiring an ultra low LLOQ

BACKGROUND FTIH studies can be challenging due to the varying dosing regimens and rapid data delivery. Chemists are asked to provide ultra-low limits of quantitation to provide an understanding of patient efficacy and safety in order to progress drug development. In a recent dermal study it became necessary to reduce the LLOQ of a small molecule drug from 50 to 1 pg/ml due to reductions in the dose and surface area of drug application. METHODOLOGY The 50-fold increase in assay sensitivity necessitated the use of a high-resolution mass spectrometer (LC-HRMS) to separate matrix interferences observed when using a unit resolution triple quadrupole MS. CONCLUSION A sensitive, robust assay was validated to support of a FTIH study using a LC-HRMS.