Manfred Zell

High-throughput quantification of drugs and their metabolites in biosamples by LC-MS/MS and CE-MS/MS: Possibilities and limitations

Off-line solid phase extraction with C18 disk plates and turbulent flow chromatography were evaluated versus on-line solid phase extraction using column-switching HPLC as sample preparation techniques for high-throughput analysis of pharmaceutical compounds and their metabolites by LC-MS/MS. Turbulent flow chromatography was found to be very straightforward in its applicaton, but the LOQs were more than fivefold higher compared with off-line or other on-line solid phase extraction methods. Solid phase extraction (SPE) on disk was found to be fast and sufficient efficient to minimize matrix effects and therefore an apprach to provide sensitive and reliable LC-MS/MS methods. Column-switching HPLC with microbore columns (0.5 mm i.d.) were used for fast analysis of a parent drug and four of its metabolites utilizing steep gradients in 1 minute. The application of CZE-MS/MS for bionalysis of pharamaceutical compounds is also discussed.

Column-switching high-performance liquid chromatography combined with ionspray tandem mass spectrometry for the simultaneous determination of the platelet inhibitor Ro 44-3888 and its pro-drug and precursor metabolite in plasma

A liquid chromatographic/mass spectrometric (LC/MS) assay was developed for the simultaneous determination of a pro-drug (Ro 48-3657), its active metabolite (platelet inhibitor, Ro 44-3888) and precursor metabolite (Ro 48-3656) in human, dog and rat plasma, utilizing on-line column-switching solid-phase extraction (SPE) for clean-up and high-performance liquid chromatography (HPLC) for separation of the analytes, with on-line detection by ionspray (pneumatically assisted electrospray) tandem mass spectrometry in the selected reaction monitoring (SRM) mode. The assay was validated for the quantification of all three analytes. The method involves protein precipitation with perchloric acid, enrichment of the analytes on a standard bore trapping column (i.d. 4.6 mm) and separation on a narrow-bore analytical column (i.d. 2 mm). Except for the plasma precipitation step, the assay was fully automated, allowing unattended operation. The lower limits of quantification were 0.20 ng ml-1 (Ro 48-3657, Ro 44-3888) and 0.50 ng ml-1 (Ro 48-3656) using a 0.5 ml plasma aliquot. The mean inter-assay precision and accuracy derived from quality control samples were 5.3% and 101%, respectively, utilizing the calibration range 0.2-200 ng ml-1. Using the unique features of column-switching HPLC combined with MS/MS, it was possible to develop the method in a short period of time. The method has been successfully applied to map complete concentration-time courses for the kinetic evaluation of the drug and its metabolites in man, dog and rat. This LC/MS assay is sensitive, specific, accurate, precise and robust.

Low picogram determination of Ro 48-6791 and its major metabolite, Ro 48-6792, in plasma with column-switching microbore high-performance liquid chromatography coupled to ion spray tandem mass spectrometry

A coupled liquid chromatography/tandem mass spectrometry assay was developed for simultaneous determination of Ro 48-6791 and its secondary amine metabolite in human plasma samples with a quantification limit for both compounds of 1 pg/mL using a 1 mL plasma aliquot. The method exploits the enhanced mass sensitivity of a microbore (300 microns i.d.) reversed-phase capillary column coupled to an ion spray probe combined with tandem mass spectrometry. A straightforward column-switching system was utilized to focus the analytes onto a microbore trapping column following solid-phase extraction of a 50 microL plasma sample extract from liquid/liquid extraction. Backflushing of the retained analytes from the trapping column onto the microbore capillary column provided the requisite high peak concentration for high sensitivity. The inter-assay precision and accuracy for Ro 48-6791 and its metabolite, at 10 pg/mL, were found to be 3.4%, and 105%, and 9.1%, and 99.9%, respectively. The calibration curves were linear over the range 1 to 1000 pg/mL. The method proved to be sufficiently rugged for analysis of samples.

Flumazenil kinetics in the elderly

SummaryIn an open design, randomised, two-way cross-over study, a single 2 mg i.v. dose and a single 30 mg oral dose of flumazenil were each administered to a group of healthy young (n=6) and elderly (n=12) volunteers (male: female 2/1). Plasma samples were collected at intervals and intact drug was assayed.Both the IV and oral doses of flumazenil were very well tolerated by both age groups and no severe or unexpected adverse effects were observed. The main complaints were dizziness and headache, mainly after oral dosing, probably due to the higher Cmax and AUC following this route of administration. After 2 mg i. v. the disposition parameters in the two age groups (elderly/young) were very similar: volume of distribution (Vss): 0.88/0.901·kg−1; total body clearance (ClPL): 0.86/0.99 l·min−1; terminal elimination half-life (t1/2β): 1.02/0.91 h. After the 30 mg oral dose the mean Cmax of 87.6 ng·ml−1 (elderly) and 78.4 ng·ml−1 (young) were generally reached within 0.5 to 1 h. In 26% (elderly) and 23% (young), the absolute bioavailability of flumazenil was very similar.It is concluded that the absorption and disposition paramters of flumazenil were not significantly affected by aging.

Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans

Abstract 1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [14C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

Determination of dalcetrapib by liquid chromatography-tandem mass spectrometry.

The cholesteryl ester transfer protein modulator dalcetrapib is currently under development for the prevention of dyslipidemia and cardiovascular disease. Dalcetrapib, a thioester, is rapidly hydrolyzed in vivo to the corresponding thiophenol which in turn is further oxidized to the dimer and mixed disulfides (where the thiophenol binds to peptides, proteins and other endogenous thiols). These forms co-exist in an oxidation-reduction equilibrium via the thiol and cannot be stabilized without influencing the equilibrium, hence specific determination of individual components, i.e., in order to distinguish between the free thiol, the disulfide dimer and mixed disulfide adducts, was not pursued for routine analysis. The individual forms were quantified collectively as dalcetrapib-thiol (dal-thiol) after reduction under basic conditions with dithiothreitol to break disulfide bonds and derivatization with N-ethylmaleimide to stabilize the free thiol. The S-methyl and S-glucuronide metabolites were determined simultaneously with dal-thiol with no effect from the derivatization procedure. Column-switching liquid chromatography-tandem mass spectrometry provided a simple, fast and robust method for analysis of human and animal plasma and human urine samples. Addition of the surfactant Tween 80 to urine prevented adsorptive compound loss. The lower limits of quantitation (LLOQ) were 5 ng/mL for dal-thiol, and 5 ng/mL for the S-methyl and 50 ng/mL for the S-glucuronide metabolites. Using stable isotope-labeled internal standards, inter- and intra-assay precisions were each <15% (<20% at LLOQ) and accuracy was between 85 and 115%. Recovery was close to 100%, and no significant matrix effect was observed.

Pharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeys.

The pharmacokinetics and excretion of carmegliptin, a novel dipeptidyl peptidase IV inhibitor, were examined in rats, dogs, and cynomolgus monkeys. Carmegliptin exhibited a moderate clearance, extensive tissue distribution, and a variable oral bioavailability of 28–174%. Due to saturation of intestinal active secretion, the area under the plasma concentration–time curve (AUC) in dogs and monkeys increased in a more than dose-proportional manner over an oral dose range of 2.5–10 mg/kg. Following oral administration of [14C]carmegliptin at 3 mg/kg, > 94% of the radioactive dose was recovered in 72-h post-dose from Wistar rats and Beagle dogs. Virtually, the entire administered radioactive dose was excreted unchanged in urine, intestinal lumen, and bile. Approximately 36%, 29%, and 19% of the dose were excreted by respective routes. Consistently, in vitro, carmegliptin was highly resistant to hepatic metabolism in all species tested. Based on in vitro studies, carmegliptin is a good substrate for Mdr1/MDR1. Breast cancer resistance protein (Bcrp) is not expected to be involved in the transport of carmegliptin since in vitro carmegliptin was not significantly transported by this transporter. The very high extravascular distribution of carmegliptin in the intestinal tissues, as demonstrated in Wistar rats and Beagle dogs, could play a significant role in its therapeutic effect.

A double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]-basimglurant and absolute bioavailability after oral administration and concomitant intravenous microdose administration of [13C6]-labeled basimglurant in humans

Abstract 1. The emerging technique of employing intravenous microdose administration of an isotope tracer concomitantly with an [14C]-labeled oral dose was used to characterize the disposition and absolute bioavailability of a novel metabotropic glutamate 5 (mGlu5) receptor antagonist under clinical development for major depressive disorder (MDD). 2. Six healthy volunteers received a single 1 mg [12C/14C]-basimglurant (2.22 MBq) oral dose and a concomitant i.v. tracer dose of 100 μg of [13C6]-basimglurant. Concentrations of [12C]-basimglurant and the stable isotope [13C6]-basimglurant were determined in plasma by a specific LC/MS-MS method. Total [14C] radioactivity was determined in whole blood, plasma, urine and feces by liquid scintillation counting. Metabolic profiling was conducted in plasma, urine, blood cell pellet and feces samples. 3. The mean absolute bioavailability after oral administration (F) of basimglurant was ∼67% (range 45.7–77.7%). The major route of [14C]-radioactivity excretion, primarily in form of metabolites, was in urine (mean recovery 73.4%), with the remainder excreted in feces (mean recovery 26.5%). The median tmax for [12C]-basimglurant after the oral administration was 0.71 h (range 0.58–1.00) and the mean terminal half-life was 77.2 ± 38.5 h. Terminal half-life for the [14C]-basimglurant was 178 h indicating presence of metabolites with a longer terminal half-life. Five metabolites were identified with M1-Glucuronide as major and the others in trace amounts. There was minimal binding of drug to RBCs. IV pharmacokinetics was characterized with a mean ± SD CL of 11.8 ± 7.4 mL/h and a Vss of 677 ± 229 L. 4. The double-tracer technique used in this study allowed to simultaneously characterize the absolute bioavailability and disposition characteristics of the new oral molecular entity in a single study.

Pharmacokinetics and Pharmacodynamics of Ro 41–3696, a Novel Nonbenzodiazepine Hypnotic

This report describes the first evaluation in humans of Ro 41–3696. Based on its preclinical profile, Ro 41–3696, a nonbenzodiazepine partial agonist at the benzodiazepine receptor, offers promising perspectives as an innovative hypnotic drug in that it does not exhibit most of the disadvantages associated with full agonists. Single oral doses of 0.1, 0.3, 1.0, 3.0, 10, and 30 mg were administered sequentially to six groups of six healthy male volunteers in a placebo‐controlled, double‐blind design. Tolerability was assessed and pharmacokinetic and pharmacodynamic measurements were conducted during a period of 28 hours after drug intake. Ro 41–3696 was well tolerated at all doses, causing no clinically relevant changes in vital signs or laboratory parameters. At doses of 10 and 30 mg there were signs of unsteady gait, indicating a central nervous system depressant effect. Pharmacokinetic analyses revealed that Ro 41–3696 was absorbed and eliminated rapidly (tmax = ∼1 hour; t1/2 = ∼4 hours). At all times plasma levels of Ro 41–3290, the desethylated derivative of Ro 41–3696, were higher than those of the parent drug (tmax and t1/2 values = ∼2 and 8 hours, respectively). Area under the curve (AUC) data indicated dose‐proportional pharmacokinetics for both Ro 41–3696 and Ro 41–3290. Performance in both a tracking and a memory search test was significantly affected by doses of 10 and 30 mg, and long‐term memory, as assessed by a word learning and recall test, was slightly impaired at these doses. The results of this study support the initiation of therapeutic efficacy studies with Ro 41–3696 in doses up to ∼5 mg and further exploration of the characteristics of Ro 41–3290.

Profiling of dalcetrapib metabolites in human plasma by accelerator mass spectrometry and investigation of the free phenothiol by derivatisation with methylacrylate

&NA; Dalcetrapib, a thioester prodrug, undergoes rapid and complete conversion in vivo to its phenothiol metabolite M1 which exerts the targeted pharmacological response in human. In clinical studies, M1 has been quantified together with its dimer and mixed disulfide species that represent the ‘dalcetrapib active form’ in plasma. In this article, we describe the determination of the free phenothiol M1 by derivatisation with methylacrylate as a percentage of ‘dalcetrapib active form’. Pharmacokinetic profiles of M1 after oral administration of dalcetrapib to humans could be established, underscoring the validity to use a composite measure of ‘dalcetrapib active form’ as a surrogate marker for pharmacodynamic evaluations. ‘Dalcetrapib active form’ and M1 made up 8.9% and 3.6% of total drug‐related material, respectively. In addition, complete metabolite profiling of 14C‐labeled dalcetrapib was conducted after two‐dimensional HPLC using fast fractionation into 384‐well plates and ultrasensitive determination of the 14C‐content by accelerator mass spectrometry. M1 underwent further biotransformation to its S‐methyl metabolite M3, which was further oxidized to its sulfoxide and sulfone. Another metabolic pathway was the formation of the S‐glucuronide. All of these species underwent further oxidation in the ethylbutyl cyclohexyl moiety leading to a multitude of hydroxyl and keto metabolites undergoing further conjugation to O‐glucuronides. More than 80 metabolites were identified, demonstrating extensive metabolism. However, it was unambiguously demonstrated that none of these metabolites were major according to the MIST guideline (exceeding 10% of drug related material in circulation). The combination of accelerator mass spectrometry with HPLC together with high resolution mass spectrometry allowed for structural characterization of the most relevant human metabolites.