JA0021 A quantitative determination of fluorochloridone in rat plasma by UPLC-MS/MS method: application to a pharmacokinetic study

: A precise, high-throughput and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC-MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 →165.2 for IS. This method was well validated with good linear response (r 2 > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra- and inter-day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration.

PO0017 Dispersive Pipette Extraction for Automated Enrichment of IGF-1 from Serum

Accurate and robust quantification of insulin-like growth factor 1 (IGF-1) from serum using mass spectrometry (MS) is challenging due to its high complexity and dynamic range. Low cost,high throughput enrichment method for such low abundant proteins from the serum is required to enrich target proteins prior to MS analysis. We report a high-throughput sample reparation method using reverse phase micro-solid phase extraction enrichment on Microlab® NIMBUS® robotic liquid handling system from Hamilton Robotics and demonstrated over 71% recovery from serum for 96 samples in less than 30 minutes. IGF-1 levels in serum correlates well to other published methods that involve longer processing times.

PO0025 Modular Automated Sample Extractions using IMCStips™on Hamilton® Microlab STAR™for High Throughput Protein Purifications

PO0024 Integrating Complex Multi-Method Workflows on a Single Hamilton®Microlab STAR™Workstation using INtip™Chemistries

PO0023 Automating Multi-Attribute Monitoring Process for At-Line Monitoring of Biotherapeutics

Protein purification is a multi-step process that incorporates various resins and consumables to achieve the desired outcome.For instance, recombinant protein purification is typically achieved using an affinity resin such as Ni-NTA followed by bufferexchange to remove the excess salts. Other affinity-based purification methods are also similar in that the IgG purified
with Protein A or G utilize basic buffer to neutralize the acidic elution, but the final storage buffer may require alternativeformulations. Here, we integrated Hamilton Microlab STAR automated liquid handling system with IMCStips to achieve highthroughput protein purification followed by multiple workflow steps to alleviate switching equipment or consumables foreach step. The first workflow is the purification of the recombinant protein by Ni-NTA, followed by buffer exchange on SizeX150 to remove the excess imidazole salts. The purified protein is then aliquoted for protein quantification. The workflowdemonstrates turn-key approach from cell lysate to quantified, purified proteins within less than 2 hours per batch. Thesecond workflow is the characterization of IgG afer Protein A purification followed by denaturation, reduction, alkylation, buffer exchange on SizeX followed by trypsin digestion for multi-attribute monitoring. The third is the reverse phase peptidedesalting and phosphopeptide enrichment on a single workflow. And the last workflow is the use of streptavidin beads
inside the pipette tip to capture biotinylated anti-Fc antibody, followed by pull-down of Avastin in mouse serum. The elutedsample is then reduced, alkylated, trypsin digested for quantifying biotherapeutic antibody in mouse sera. These integratedworkflows with IMCStips demonstrate purification of different proteins using different INtip™ chemistries and the flexibility ofthe Hamilton Microlab STAR to achieve a variety of processes on a single platform to achieve high throughput workflows withminimal human intervention.

PO0021 Selective Enrichment of Peptides by Utilizing Dispersive Pipette Extraction on AutomatedLiquid Handler for High Throughput Discovery and Processing

According to a database established from human hemofiltrate, approximately 5000 different peptides were recorded. Over 95% of the detected masses were smaller than 15,000 Da and a small percentage (7%) of the detected masses represented sequences frompeptide hormones, growth factors and cytokines. Tese low molecular weight (LMW) peptides/proteins are attractive targets for monitoring human health. Here, we demonstratea proof of concept size exclusion chromatography technique using INtip, dispersive pipetteextraction chemistries (IMCStips™) for rapid isolation of LMW peptides(Bradykinin and Angiotensin I) by using various pore sizes ranging from 30 Å to 60 Å.

PO0022 Affinity-based Dispersive Pipette Extraction for Automated Purification

Affinity-based extractions in complex matrixes are typically time-consuming due to the la-bor intensive work of hands on pipetting and centrifugation. A typical spin column affinity extraction takes an hour to bind the sample and requires excess buffer solutions towash off non-specific binding proteins using multiple centrifugation steps. A quick, highthroughput affinity-based enrichment method for target proteins is desired to allow forquicker screening of samples. We report a high-throughput sample preparation method us-ing antibody and his-tag based enrichment on a robotic liquid handling system with disper-sive pipette extraction. Specifically, Protein A and cobalt-IMAC resins were used for IgGenrichment from human serum and his-tagged proteins from cell lysates, respectively. Te Protein A extractions were compared side-by-side with a traditional spin-column based extraction protocol. Two different his-tagged proteins were purified fromcell lysate using the IMAC resins in tips. Tese developments allow for completely automat-ed 30 minute sample preparation leading to high-throughput sample screening and bettercompatibility with in-house automated liquid handling systems.

PO0019 Increased Throughput of INtip Affinity Purification for Larger Sample Volume

PO0018 Expanding Functionality of Automated Liquid Handling Systems by Incorporating INtip Chemistriesfor High Throughput Protein Purification

PO0020 Enhanced Recovery of Trypsin Digested Proteins Using Dispersive Pipette Extraction for Downstream Proteomic Analysis

INTRODUCTION
For clinical proteomics, targeted peptide extraction and desalting efficiencies areessential to obtaining high quality mass spectra data. In this study, we report anovel peptide purification method usingIMCStips™,a disposable pipetteextraction tip with sorbent loosely contained inside a pipette tip. Using bovineserum albumin as a model protein and four other human serum proteins,trypsin-digestedsamples were readilydesaltedusingIMCStips™ with reversephase resin. The purificationimproved signalintensities, signal-to-noise ratiosand sequencecoverage in comparison to other commercially available tips.Sincethis method can be effortlessly adapted to an automated online format, weenvision it to be applied in the high throughput clinical proteomics application.