Christopher Holliman

Utilization of hydrophilic-interaction LC to minimize matrix effects caused by phospholipids

BACKGROUND In bioanalysis, phospholipids may affect the precision and accuracy of LC-MS/MS methods and compromise the quality of the results, especially when samples in complex biomatrices are extracted by protein precipitation techniques. RESULTS It was found that the retentive behavior of both common pharmaceuticals and physiologically relevant phospholipids under bare silica hydrophilic-interaction LC (HILIC) is more predictable than under reversed-phase conditions. In particular, the retention time of phospholipids was not significantly affected by varying the salt and acid modifiers in the mobile phases, but common pharmaceuticals can be shifted away from these phospholipid interferences through mobile phase modifiers. Several mass spectrometric techniques were applied to confirm this finding. CONCLUSION HILIC chromatography is a valued tool in the development of robust bioanalytical assays with minimal and predictable phospholipid interferences. Furthermore, addition of a small amount of ion-pairing additives can reliably move pharmaceutical compounds away from these suppressive regions.

Application of the dried spot sampling technique for rat cerebrospinal fluid sample collection and analysis

Dried blood spotting (DBS) sample collection is gaining favor in the pharmaceutical industry due to benefits that include reduced animal usage and easier sample shipment and storage when compared to traditional plasma collection/analysis. The applicability of the DBS card to alternate, limited-volume, matrices has not been as fully characterized as their use with whole blood. In this paper we explored the application of the DBS sample collection technique to rat cerebrospinal fluid (CSF). A reverse phase HPLC-MS/MS method was developed and characterized for the quantitative bioanalysis of the α7 neutonal nicotinic acetylcholine receptor agonist PHA-00543613 in CSF using the dried spot sampling technique. The characterized assay and dried spot sampling technique was employed to analyze serially collected in vivo rat CSF samples after a single 4mg/kg dose of PHA-00543613 in CSF-cannulated rats. The DBS strategy enabled the collection of more timepoints and produced comparable exposure results to those obtained by the collection and analysis of liquid CSF samples but notably with eight less animals.

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies

Pregabalin (Lyrica) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a gamma-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.

Small Molecule Specific Run Acceptance, Specific Assay Operation, and Chromatographic Run Quality Assessment: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Teams

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

A multiplex HRMS assay for quantifying selected human plasma bile acids as candidate OATP biomarkers

AIM Selected bile acids (BAs) in plasma have been proposed as endogenous probes for assessing drug-drug interactions involving hepatic drug transporters such as the organic anion-transporting polypeptides (OATP1B1 and OATP1B3). MATERIALS & METHODS Plasma extracts were analyzed for selected BAs using a triple TOF API6600 high-resolution mass spectrometer. RESULTS Glycodeoxycholic acid 3-sulfate, glycochenodeoxycholic acid 3-sulfate, glycodeoxycholic acid 3-O-β-glucuronide and glycochenodeoxycholic acid 3-O-β-glucuronide are presented as potential OATP1B1/3 biomarkers. CONCLUSION Six BAs are quantified in human plasma using a multiplexed high-resolution mass spectrometry method. Glycodeoxycholic acid 3-sulfate and glycodeoxycholic acid 3-O-β-glucuronide are proposed as potential biomarkers based on observed four- to fivefold increase in plasma AUC (vs placebo), following administration of a compound known to present as an OATP1B1/3 inhibitor in vitro.