LC-MS/MS simultaneous quantification of apomorphine and its major metabolites in human plasma: Application to clinical comparative bioavailability evaluation for the apomorphine sublingual film and a subcutaneous product.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantification of apomorphine and its metabolites apomorphine sulfate and norapomorphine in human plasma for supporting clinical development of a novel apomorphine sublingual thin film (APL) for the treatment of Parkinson's disease. Analytes and internal standards (IS) were extracted from human plasma by Oasis HLB SPE cartridge, followed by a reversed phase LC-MS/MS analysis using multiple reaction monitoring (MRM) in positive mode (m/z 268 → 237 for apomorphine, 348 → 237 for apomorphine sulfate, and 348 → 237 for norapomorphine). Stable isotope-labeled compounds were used as IS for respective analytes. The validated curve ranges were 0.02-20 ng/mL, 10-1000 ng/mL, and 0.5-20 ng/mL for apomorphine, apomorphine sulfate and norapomorphine, respectively. Extraction recoveries were found to be 73.4 % (apomorphine), 81.1 % (apomorphine sulfate), and 58.6 % (norapomorphine). Established long-term plasma frozen storage stabilities were 504 days at -20 °C and276 days at -60 °C, respectively. The method has been successfully used for analyzing pharmacokinetics (PK) samples collected from a comparative bioavailability study of APL and the marketed apomorphine subcutaneous (s.c.) product Apo-go®. The results demonstrated that the 15-mg APL film administrated via sublingual produced comparable PK characteristics of apomorphine when compared to the commercial product Apo-go (2-mg) via s.c. administration, hence establishing the dose regimen for this sublingual formulation. It was also noticed that the sublingual 15-mg APL film produced a significantly higher apomorphine sulfate metabolite level than the 2-mg s.c. Apo-go, and both treatments yielded a negligible level of norapomorphine metabolite in humans.

Validation of a sensitive and specific LC-MS/MS method and application to evaluate the systemic exposure and bioavailability of glycopyrrolate via different drug delivery approaches/devices.

A specific and sensitive LC-MS/MS method using d3 -glycopyrrolate as the internal standard (IS) was developed for the quantitative determination of glycopyrrolate (GLY) in human plasma over a concentration range of 4.00-2000 pg/ml. The GLY and IS were extracted using a solid-phase extraction cartridge, then eluted with 0.5% formic acid in 70:30 acetonitrile-water. The eluate was directly injected into an Agilent Pursuit 5PFP column for analysis using an isocratic mobile phase of 50:50 A:B at a flow-rate of 0.500 ml/min (A, 10 mm ammonium acetate in 1% formic acid; B, methanol). The MS detection was in positive mode by monitoring m/z 318.3 → 116.1 (GLY) and 321.3 → 119.1 (IS). The method validation showed the linearity of r2 ≥ 0.9960, intra-/inter-run precisions of ≤11.1% coefficient of variation and accuracies ranging from -2.5 to 12.8% relative error for all levels of quality control samples. This method was successfully employed to support a clinical study to compare absorption and bioavailability of GLY administered by a Magnair® eFlow nebulizer to a Seebri® Breezhaler® with/without a charcoal blockade of gastric absorption. By comparison with intravenous administration, respective bioavailabilities of ~15% for GLY/Magnair and ~22% for the Seebri Breezhaler were found. The bioanalytical reliability was also demonstrated by satisfactory incurred sample reanalysis performance.

Structural Characterization and Disulfide Assignment of Spider Peptide Phα1ß by Mass Spectrometry.

Native Phα1ß is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1ß mimics the effects of the native Phα1ß. Because of this, it has been suggested that Phα1ß may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1ß is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1ß peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1ß peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1ß peptide. These experiments provide essential structural information about Phα1ß and may assist in providing insight into the peptide's analgesic effect with very low side effects. Graphical Abstract ᅟ.