Mass spectrometry for small molecule pharmaceutical product development: a review.

Developing a pharmaceutical product has become increasingly difficult and expensive. With an emphasis on developing project knowledge at an earlier stage in development, the use of information-rich technologies (particularly MS) has continued to expand throughout product development. Continued improvements in LC/MS technology have widened the scope of utilizing MS methods for performing both qualitative and quantitative applications within product development. This review describes a multi-tiered MS strategy designed to enhance and accelerate the identification and profiling of both process- and degradation-related impurities in either the active pharmaceutical ingredient (API) or formulated product. Such impurities can be formed either during chemical synthesis, formulation, or during storage. This review provides an overview of a variety of orthogonal-mass spectrometric methodologies, namely GC/MS, LC/MS, and ICP-MS, in support of product development. This review is not meant to be all inclusive; however, it has been written to highlight the increasing use of hyphenated MS techniques within the pharmaceutical development area.

Identification and counting of carbonyl and hydroxyl functionalities in protonated bifunctional analytes by using solution derivatization prior to mass spectrometric analysis via ion-molecule reactions.

A mass spectrometric method has been developed for the identification of carbonyl and hydroxyl functional groups, as well as for counting the functional groups, in previously unknown protonated bifunctional oxygen-containing analytes. This method utilizes solution reduction before mass spectrometric analysis to convert the carbonyl groups to hydroxyl groups. Gas-phase ion-molecule reactions of the protonated reduced analytes with neutral trimethylborate (TMB) in a FT-ICR mass spectrometer give diagnostic product ions. The reaction sequence likely involves three consecutive steps, proton abstraction from the protonated analyte by TMB, addition of the neutral analyte to the boron reagent, and elimination of a neutral methanol molecule. The number of methanol molecules eliminated upon reactions with TMB reveals the number of hydroxyl groups in the analyte. Comparison of the reactions of the original and reduced analytes reveals the presence and number of carbonyl and hydroxyl groups in the analyte.

Identification of aliphatic and aromatic tertiary N-oxide functionalities in protonated analytes via ion/molecule and dissociation reactions in an FT-ICR mass spectrometer.

A mass spectrometric method is presented for the identification of compounds that contain the aliphatic or aromatic N-oxide functional group. This method utilizes gas-phase ion/molecule reactions of tri(dimethylamino)borane (TDMAB), which rapidly derivatizes protonated aliphatic and aromatic tertiary N-oxides, amides, and some amines via loss of dimethylamine in a Fourier transform ion cyclotron resonance mass spectrometer. The mechanism involves proton transfer from the protonated analyte to the borane, followed by addition of the analyte to the boron center and elimination of dimethylamine. The derivatized analytes are readily identified on the basis of their m/z value which is 98 Th (thomson) greater than that of the protonated analyte, and the characteristic boron isotope patterns. SORI-CAD of the product ions (adduct-(CH3)2NH) yields different fragment ions for aliphatic tertiary N-oxides, aromatic tertiary N-oxides, amides, and pyridines. Therefore, these analytes can be identified based on their characteristic fragment ions. This method was tested by examining two drug samples, Olanzapine and Olanzapine-4' N-oxide.

Identification of the aromatic tertiary N-oxide functionality in protonated analytes via ion/molecule reactions in mass spectrometers.

A mass spectrometric method is presented for the rapid identification of compounds that contain the aromatic N-oxide functional group. This method utilizes a gas-phase ion/molecule reaction with 2-methoxypropene that yields a stable adduct for protonated aromatic tertiary N-oxides (and with one protonated nitrone) in different mass spectrometers. A variety of protonated analytes with O- or N-containing functional groups were examined to probe the selectivity of the reaction. Besides protonated aromatic tertiary N-oxides and one nitrone, only three protonated amines were found to form a stable adduct but very slowly. All the other protonated analytes, including aliphatic tertiary N-oxides, primary N-oxides, and secondary N-oxides, are unreactive toward or react predominantly by proton transfer with 2-methoxypropene.

The disposition of prasugrel, a novel thienopyridine, in humans.

Prasugrel, a prodrug, is a novel and potent inhibitor of platelet aggregation in vivo. The metabolism of prasugrel and the elimination and pharmacokinetics of its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), three inactive metabolites, and radioactivity were determined in five healthy male subjects after a single 15-mg (100 microCi) p.o. dose of [(14)C]prasugrel. Prasugrel was rapidly absorbed, and maximum plasma concentrations of radioactivity and R-138727 were achieved in 30 min, indicating rapid formation of R-138727. Prasugrel was extensively metabolized in humans, first by hydrolysis to a thiolactone, followed by ring opening to form R-138727, which was further metabolized by S-methylation and conjugation with cysteine. Total radioactivity was higher in plasma than in blood, suggesting limited penetration of prasugrel metabolites into red blood cells. Approximately 70% of the dose was excreted in the urine and 25% in the feces.

The disposition and metabolism of naveglitazar, a peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist in mice, rats, and monkeys.

Naveglitazar [LY519818; benzenepropanoic acid, alpha-methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (alpha-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [(14)C]naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at C(max), representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the R-enantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (>99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics.

Although traditionally reserved for proteomic analysis, nanoESI has found increased use for small molecule applications related to drug metabolism/pharmacokinetics (DMPK). NanoESI, which refers to ESI performed at flow rates in the range of 200 to 1000 nL/min using smaller diameter emitters (10 to 100 microm id), produces smaller droplets than conventional ESI resulting in more efficient ionization. Benefits include greater sensitivity, enhanced dynamic range, and a reduced competition for ionization. These advantages may now be harnessed largely due to the introduction of a commercial system for automated nanoESI infusion. This development in turn has allowed ADME (absorption, distribution, metabolism, and excretion) scientists to consider novel approaches to mass spectrometric analysis without direct LC interfacing. While it is freely acknowledged that nanoESI infusion is not likely to supplant LC-MS as the primary analytical platform for ADME, nanoESI infusion has been successfully applied to both quantitative (bioanalysis) and qualitative (metabolite identification) applications. This review summarizes published applications of this technology and offers a perspective on where it fits best into the DMPK laboratory.

Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry.

Imaging mass spectrometry (IMS) that utilizes matrix-assisted laser desorption/ionization (MALDI) technology can provide a molecular ex vivo view of resected organs or whole-body sections from an animal, making possible the label-free tracking of both endogenous and exogenous compounds with spatial resolution and molecular specificity. Drug distribution and, for the first time, individual metabolite distributions within whole-body tissue sections can be detected simultaneously at various time points following drug administration. IMS analysis of tissues from 8 mg/kg olanzapine dosed rats revealed temporal distribution of the drug and metabolites that correlate to previous quantitative whole-body autoradiography studies. Whole-body MALDI IMS is further extended to detecting proteins from organs present in a whole-body sagittal tissue section. This technology will significantly help advance the analysis of novel therapeutics and may provide deeper insight into therapeutic and toxicological processes, revealing at the molecular level the cause of efficacy or side effects often associated with drug administration.

Application of biocatalysis to drug metabolism: preparation of mammalian metabolites of a biaryl-bis-sulfonamide AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor potentiator using Actinoplanes missouriensis.

LY451395 (2-propanesulfonamide, N-[(2R)-2-[4'-[2-[methylsulfonyl)amino]ethyl][1,1'-biphenyl]-4-yl]propyl]-) is a potent and highly selective potentiator of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. It is a biaryl-bis-sulfonamide and is known to be highly metabolized in preclinical species. In those metabolism studies, the metabolite structures were proposed exclusively by the analysis of mass spectrometric data. Although mass spectrometry is clearly a technique of choice for rapid identification of drug metabolites, occasionally, nuclear magnetic resonance spectroscopy is required to unambiguously assign and characterize, particularly, the regio- and stereochemistry of metabolic changes. Nuclear magnetic resonance spectroscopy, in general, is less sensitive than other detection methods and demands several micrograms of material for the analysis. To support full structure characterization of metabolites by NMR, in this study we demonstrated the application of a microbial-based surrogate biocatalytic system to produce sufficient amounts of the mammalian metabolites of LY451395. The results revealed that incubation of LY451395 with Actinoplanes missouriensis NRRL B3342 generated several metabolites that were previously detected in the in vivo metabolism studies of the preclinical species. Subsequent large-scale bioconversion resulted in the isolation of seven mammalian metabolites in milligram quantities for structural characterization by nuclear magnetic resonance spectroscopy. Furthermore, a selected group of metabolites generated from the microbial conversion served as analytical standards to monitor and quantify drug metabolites during clinical investigations.

Pergolide: multiple-dose pharmacokinetics in patients with mild to moderate Parkinson disease.

The primary objective of this study was to describe the pharmacokinetics of oral pergolide in patients with mild to moderate Parkinson disease using a new high-performance liquid chromatography-tandem mass spectrometry assay. A secondary objective was to investigate the relationship between plasma concentrations and efficacy. Fourteen patients with a diagnosis of Parkinson disease completed this multicenter, open-label, dose-escalating study. Pergolide was administered for 58 days, using increasing daily doses from 0.05 mg daily up to 1 mg three times daily and then tapering the dose. The steady-state pharmacokinetic profile and motor score were determined at dose levels of 0.25, 0.5, and 1 mg three times a day and during elimination after the last dose. Pergolide was absorbed with a median time to maximum concentration of 2 to 3 hours across the dose range. Systemic exposure appeared to increase proportionally with dose over the range of 0.25 to 1 mg three times daily within a patient, but there was a large variability in exposures between patients (interpatient coefficients of variation were 56.4% for the area under the curve). Pergolide was widely distributed (volume of distribution, approximately 14,000 L) and was eliminated with a mean half-life of 21 hours. Motor scores improved as both peak plasma pergolide concentrations and exposure increased. No unexpected safety concerns were identified. Pergolide is absorbed relatively quickly into the systemic circulation, has a large apparent volume of distribution, and has a relatively long half-life (mean, 21 hours). This prolonged half-life is of particular interest, given the current hypothesis that more continuous dopaminergic receptor stimulation may reduce motor complications in patients with Parkinson disease.