Drug monitoring by volumetric absorptive microsampling: method development considerations to mitigate hematocrit effects.

AIM: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. METHODOLOGY: After accurate sampling of 10 µl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. CONCLUSION: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

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.

Application of a Stable Isotope Approach to Evaluate Impact of Changes in Manufacturing Parameters for an Immediate-Release Tablet.

There is continued emphasis from the various worldwide regulatory agencies to ensure that the pharmaceutical industry fully understands the products they are developing. This emphasis is seen via development of quality-by-design (QbD) publications and guidelines generated by the International Committee on Harmonization. The challenge to meet these expectations is primarily associated with the generation of in vivo data (eg, pharmacokinetic data) that is resource intensive. A technique reducing the resources needed to generate this in vivo data permits a more extensive application of QbD principles. This paper presents the application of stable isotopes in pharmacokinetic studies. The data show that the use of stable isotopes can significantly reduce the number of subjects required for a study. This reduction in subjects thus translates into a significant reduction in resources and time needed to generate the required in vivo data to support QbD.

Camphanic acid chloride: a powerful derivatization reagent for stereoisomeric separation and its DMPK applications.

BACKGROUND: Camphanic acid chloride has proven to be an efficient chiral derivatization reagent for determination of stereoisomers. RESULTS: The utility of chemical derivatization of various stereoisomers containing hydroxy functional groups with camphanic acid chloride in the presence or absence of a base is highlighted. This procedure is shown to be relatively simple, fast and a cost-effective method of separating racemic drugs and stereoisomeric metabolites in biological matrices. Camphanic derivatives contain two additional chirogenic centers, which are found to enhance stereoisomeric separation on both traditional and chiral stationary phases. CONCLUSION: Four methodologies described herein for separation of multiple stereoisomers in biological samples confirm camphanic acid chloride to be a powerful chiral reagent for stereoisomeric resolution for drug metabolism and PK applications.

An integrated ceramic, micro-fluidic device for the LC/MS/MS analysis of pharmaceuticals in plasma.

An integrated capillary scale (300 µm id) ceramic microfluidic LC system combined with MS/MS has been successfully employed for the quantitative analysis of pharmaceutical compounds in human plasma. The capillary ceramic microfluidic LC/MS/MS system showed an approximate 20-fold (range 11-38-fold) increase in sensitivity compared with a standard 2.1 mm scale UPLC/MS/MS system for a broad range of analytes. The loading capacity of the devices capillary separations channel allowed injection of 2 µL of an aqueous solution, and up to 1.2 µL of a typical protein-precipitated plasma sample, onto the reversed-phase chromatography system. The system also showed excellent chromatographic performance and robustness, with no deleterious effects on the chromatography observed over the course of 1000 injections of protein-precipitated plasma. The ability of the ceramic microfluidic LC/MS/MS system to deliver this level of sensitivity and performance enables the routine quantification of pharmaceutical compounds from small format samples, such as those obtained by dried blood spot or other blood microsampling approaches, to be performed.

Evaluation of bioequivalence of five 0.1 mg dutasteride capsules compared to one 0.5 mg dutasteride capsule: a randomized study in healthy male volunteers.

OBJECTIVE: To evaluate the bioequivalence of five 0.1 mg dutasteride capsules to one 0.5 mg dutasteride capsule in healthy adult male subjects under fasting conditions. METHODS: This was a single-center, open-label, randomized, single dose, two-way cross-over study (ClinicalTrials.gov identifier NCT01929330). Thirty-six healthy male subjects aged 18-65 years received 5 × 0.1 mg dutasteride softgel capsules and 1 × 0.5 mg dutasteride softgel capsule in a randomized order, with a minimum washout of 28 days between each drug administration. Serial blood samples were collected for the measurement of serum dutasteride concentrations by a validated HPLC-MS/MS method. Dutasteride pharmacokinetic parameters were calculated using non-compartmental analysis. Maximum concentration (Cmax) and area under the concentration-time curve to the last quantifiable concentration (AUC[0-t]) were compared between treatments. Safety and tolerability were monitored throughout the study. RESULTS: Five 0.1 mg dutasteride capsules were demonstrated to be bioequivalent to 1 × 0.5 mg dutasteride capsule, as the 90% confidence intervals for Cmax and AUC were within the accepted bioequivalence range of 0.80-1.25. The geometric least squares means ratios and associated 90% confidence intervals for 5 × 0.1 mg capsules vs 1 × 0.5 mg capsule were 1.01 (0.97-1.05) for Cmax and 0.91 (0.84-1.00) for AUC(0-t). Adverse events (AEs) were reported for 42% (15/36) and 36% (12/33) of subjects in the 5 × 0.1 mg and 1 × 0.5 mg dosing sessions, respectively. The most frequent AE for both treatments was headache. No subject had a serious AE. CONCLUSIONS: Five 0.1 mg dutasteride capsules were shown to be bioequivalent to one 0.5 mg dutasteride capsule in healthy adult male subjects under fasted conditions, suggesting that the two dose strengths can be interchanged. Both treatments were generally well tolerated in healthy male subjects.

Investigation of metabolism and disposition of GSK1322322, a peptidase deformylase inhibitor, in healthy humans using the entero-test for biliary sampling.

GSK1322322 (N-((R)-2-(cyclopentylmethyl)-3-(2-(5-fluoro-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-methylpyrimidin-4-yl)hydrazinyl)-3-oxopropyl)-N-hydroxy-formamide) is an antibiotic in development by GlaxoSmithKline. In this study, we investigated the metabolism and disposition of [(14)C]GSK1322322 in healthy humans and demonstrated the utility of the Entero-Test in a human radiolabel study. We successfully collected bile from five men using this easy-to-use device after single i.v. (1000 mg) or oral administration (1200 mg in a solution) of [(14)C]GSK1322322. GSK1322322 had low plasma clearance (23.6 liters/hour) with a terminal elimination half-life of ∼4 hours after i.v. administration. After oral administration, GSK1322322 was readily and almost completely absorbed (time of maximal concentration of 0.5 hour; bioavailability 97%). GSK1322322 predominated in the systemic circulation (>64% of total plasma radioactivity). An O-glucuronide of GSK1322322 (M9) circulated at levels between 10% and 15% of plasma radioactivity and was pharmacologically inactive. Humans eliminated the radioactive dose in urine and feces at equal proportions after both i.v. and oral doses (∼45%-48% each). Urine contained mostly unchanged GSK1322322, accounting for 30% of the dose. Bile contained mostly M9, indicating that glucuronidation was likely a major pathway in humans (up to 30% of total dose). In contrast, M9 was found in low amounts in feces, indicating its instability in the gastrointestinal tract. Therefore, without the Entero-Test bile data, the contribution of glucuronidation would have been notably underestimated. An unusual N-dehydroxylated metabolite (a secondary amide) of GSK1322322 was observed primarily in the feces and was most likely formed by gut microbes.

Penetration of GSK1322322 into epithelial lining fluid and alveolar macrophages as determined by bronchoalveolar lavage.

GSK1322322 is a potent peptide deformylase inhibitor with in vitro and in vivo activity against multidrug-resistant skin and respiratory pathogens. This report provides plasma and intrapulmonary pharmacokinetics, safety, and tolerability of GSK1322322 after repeat (twice daily intravenous dosing for 4 days) dosing at 1,500 mg. Plasma samples were collected over the last 12-hour dosing interval of repeat dosing following the day 4 morning dose (the last dose). Bronchoalveolar lavage samples were collected once in each subject, either before or at 2 or 6 h after the last intravenous dose. Plasma area under the concentration-time curve (AUC0-τ) was 66.7 µg · h/ml, and maximum concentration of drug in serum (Cmax) was 25.4 µg/ml following repeat doses of intravenous GSK1322322. The time course of epithelial lining fluid (ELF) and alveolar macrophages (AM) mirrored the plasma concentration-time profile. The AUC0-τ for ELF and AM were 78.9 µg · h/ml and 169 µg · h/ml, respectively. The AUC0-τ ratios of ELF and AM to total plasma were 1.2 and 2.5, respectively. These ratios increased to 3.5 and 7.4, respectively, when unbound plasma was considered. These results are supportive of GSK1322322 as a potential antimicrobial agent for the treatment of lower respiratory tract bacterial infections caused by susceptible pathogens. (This study has been registered at ClinicalTrials.gov under registration number NCT01610388.).

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 of a sensitive and selective LC-MS/MS method for the determination of urea in human epithelial lining fluid.

A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20µL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8µm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78-103.78µg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20µL) and a run time of 1.5min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution.

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.

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.

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 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.

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.

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.

Development of a sensitive and selective LC-MS/MS method for the determination of alpha-fluoro-beta-alanine, 5-fluorouracil and capecitabine in human plasma.

A sensitive and selective quantitative method to determine alpha-fluoro-beta-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 microL) has been developed and validated. First, 5-FU and Cape were extracted by liquid-liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column "Primesep D" (2.1 mm x 50 mm; 5 microm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 microm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10-10,000, 5-5000, and 1-1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.

A semi-automated 96-well plate method for the simultaneous determination of oral contraceptives concentrations in human plasma using ultra performance liquid chromatography coupled with tandem mass spectrometry.

Two semi-automated, relatively high throughput methods using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) were developed for the simultaneous determination of ethinyl estradiol (EE) in combination with either 19-norethindrone (NE) or levonorgestrel (LN) in human plasma. Using 300 microL plasma, the methods were validated over the concentration ranges of 0.01-2 ng/mL and 0.1-20 ng/mL for EE and NE (or LN), respectively. The existing methods for the determination of the oral contraceptives in human plasma require large volumes of plasma (> or =500 microL), and sample extraction is labor-intensive. The LC run time is at least 6 min, enabling analysis of only about 100 samples a day. In the present work the throughput was greatly improved by employing a semi-automated sample preparation process involving liquid-liquid extraction and derivatization with dansyl chloride followed by UPLC separation on a small particle size column achieving a run time of 2.7 min. The validation and actual sample analysis results show that both methods are rugged, precise, accurate, and well suitable to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in a day.

Trapping 4-fluorobenzyl chloride in human plasma with chemical derivatization followed by quantitative bioanalysis using high-performance liquid chromatography/tandem mass spectrometry.

A quantitative bioanalytical method involving chemical derivatization, solid phase extraction (SPE) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was developed for the determination of 4-fluorobenzyl chloride (4FBCl) in human plasma. 4FBCl is a volatile and reactive molecule that is very unstable in human plasma. In order to stabilize 4FBCl in plasma samples prior to storage, 4-dimethylaminopyridine (DMAP) was added, forming a stable quaternary amine salt derivative. A three-step weak cation-exchange SPE procedure was then employed to remove excess DMAP. The plasma extracts were analyzed by HPLC/MS/MS using a TurboIonspray interface and multiple reaction monitoring. Unlike 4FBCl, the quaternary amine derivative shows excellent sensitivity in electrospray mass spectrometry. The method was validated over a concentration range of 0.5-500 ng/mL using 45 microL of plasma. The maximum within-run and between-run precision observed in a three-run validation for quality control (QC) samples was 12.5 and 7.6%, respectively. The maximum percentage bias observed at all QC sample concentrations was 11.9%. The method has proven to be robust and compatible with high-throughput bioanalysis.