Hermes Licea-Perez

Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5α-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism

A highly sensitive and selective quantitative method to accurately determine testosterone (Te) and 5alpha-dihydrotestosterone (DHT) in human serum is crucial to the success of Te replacement therapy for hypogonadism. To this end we have developed and validated a semi-automated and relatively high-throughput method in a 96-well plate format using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) for the simultaneous determination of Te and DHT in human serum. Te and DHT along with the internal standards [(2)H(3)]-Te and [(2)H(3)]-DHT were extracted from 300 microL of human serum by liquid-liquid extraction using methyl tertiary-butyl ether (MTBE), followed by derivatization with 2,3-pyridinedicarboxylic anhydride and solid-phase extraction for sample clean up. A novel chemical derivatization approach using 2,3-pyridinedicarboxylic anhydride was employed to achieve the MS sensitivity and selectivity required for DHT. Baseline separation of Te and DHT derivatives from endogenous steroid derivatives was achieved using UPLC technology on a C18 stationary-phase column with 1.7 microm particle size. The validity of using double charcoal-stripped female human serum as surrogate matrix for preparation of calibration standards was demonstrated through standard addition experiments. The method was validated over the concentration ranges of 0.2-40 ng/mL for Te and 0.01-2 ng/mL for DHT. The validation and study sample analysis results 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.

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.

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.

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.

Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activity in vivo

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography-mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid-liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05-50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.

Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC–MS/MS

A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5-5000 ng/mL and 3-3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC-MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min.

Mass spectrometric characterization of a prolyl hydroxylase inhibitor GSK1278863, its bishydroxylated metabolite, and its implementation into routine doping controls

Drug candidates, which have the potential of enhancing athletic performance represent a risk of being misused in elite sport. Therefore, there is a need for early consideration by anti-doping authorities and implementation into sports drug testing programmes. The hypoxia-inducible factor (HIF) or prolyl hydroxylase inhibitor (PHI) GSK1278863 represents an advanced candidate of an emerging class of therapeutics that possess substantial potential for abuse in sport due to their capability to stimulate the biogenesis of erythrocytes and, consequently, the individuals oxygen transport capacity. A thorough characterization of such analytes by technologies predominantly used for doping control purposes and the subsequent implementation of the active drug and/or its main urinary metabolite(s) are vital for comprehensive, preventive, and efficient anti-doping work. In the present study, the HIF PHI drug candidate GSK1278863 (comprising a 6-hydroxypyrimidine-2,4-dione nucleus) and its bishydroxylated metabolite M2 (GSK2391220A) were studied regarding their mass spectrometric behaviour under electrospray ionization (ESI-MS/MS) conditions. Synthesized reference materials were used to elucidate dissociation pathways by means of quadrupole/time-of-flight high resolution/high accuracy tandem mass spectrometry, and their detection from spiked urine and elimination study urine samples under routine doping control conditions was established using liquid chromatography-electrospray ionization-tandem mass spectrometry with direct injection. Dissociation pathways to diagnostic product ions of GSK1278863 (e.g. m/z 291, 223, and 122) were proposed as substantiated by determined elemental compositions and MS(n) experiments as well as comparison to spectra of the bishydroxylated analogue M2. An analytical assay based on direct urine injection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of GSK1278863 in combination with its bishydroxylated metabolite M2. Validation parameters including limit of detection (0.5-1 ng/mL), linearity, specificity, ion suppression/enhancement (<10%), intra- and inter-day precision (6-22%) were determined, demonstrating the fitness-for-purpose of the assay for doping control screening of urine samples for the presence of the drug candidate and its main metabolite and for expanding current anti-doping efforts to this new class of therapeutics. However, administration study urine sample analysis suggested the use of M2 rather than the intact drug due to extensive metabolic conversion. Copyright

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.

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.

New insights into supercritical fluid chromatography for chiral separations

Bioanalysis of stereoisomers represents a great challenge, as chromatographic separation is not feasible using conventional reversed or normal-phase chromatography. Supercritical fluid chromatography in combination with chiral stationary phases has proven to be a great tool for chiral resolution, alleviating some of the challenges associated with bioanalysis of stereoisomers. This technical note describes an alternative to the traditional SFC instrument configuration without the use of a back pressure regulator (BPR) and makeup pump; thereby simplifying its usage and improving the robustness and quality of measurements. This alternative configuration has been successfully employed in our laboratory for multiple chiral applications; in particular for the separation of cis/trans geometric stereoisomers. Four case studies are presented – one of which was validated demonstrating the robustness of the technology for bioanalytical applications.