Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

Internal standardization plays a critical role in the performance of a bioanalytical method. There has been a tremendous increase in the popularity of using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for quantitative bioanalysis of protein molecules. Protein, being too large to be directly analyzed by LC-MS/MS, is proteolyzed and a characteristic peptide is used as a surrogate analyte for quantification. Internal standardization in small molecules’ analysis is straightforward, i.e., either a stable labeled isotope (SIL) form of the analyte or a structural analogue is used. As protein quantification involves protein digestion to yield peptides, there are more options for internal standardization. Currently, internal standard selection is based on the availability of the internal standards and the sample preparation workflow. A SIL-form of the analyte protein is the ideal internal standard. However, its use is limited due to cost and commercial availability. Alternatively, a SIL form the surrogate peptide analyte or a cleavable SIL-peptide can be used as an IS. For preclinical bioanalysis of humanized IgG antibody-based drugs, a universal SIL analogue protein has been effectively used as an internal standard. This chapter focuses on internal standardization for the quantitative analysis of proteins, such as biotherapeutics and biomarkers, using LC-MS/MS

Quantitative Analysis of Multiple Charged Large Molecules in Human or Rat Plasma Using Liquid Chromatography Tandem Mass Spectrometry

Immunoassays have traditionally been employed for the determination of plasma concentration-time profiles for pharmacokinetic studies of therapeutic proteins and peptides. These ligand binding assays have high sensitivity but require significant time for antibody generation (1 to 2 years) for assay development. Despite high sensitivity, these assays suffer from cross-reactivity that can lead to inaccurate results. As an alternative to immunoassays, this dissertation was focused on the development and validation of assays that can be used for quantitative analysis of peptides or proteins in plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two approaches were considered for measurement of proteins and peptides fortified in plasma. The first approach involved employing signature peptides as quantitative surrogates of a target protein. This approach is a multistep process that includes: computer simulated (in silico) peptide predictions, protein purification, proteolytic digestion, peptide purification, and ultimately mass spectrometry. Signature peptides were determined through in silico peptide predictions and iterative tuning processes to represent Amevive® (Alefacept), a therapeutic for psoriasis, for quantification in human plasma. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with optimized pH and heat conditions followed by enzymatic digestion, dilution, and filtration. Combining selective precipitation and protein analogue internal standard lead to a method validated according to current FDA guidelines and achieved a linear range (250-10,000 ng/mL) suitable for monitoring the therapeutic levels of Alefacept (500 -6000 ng/mL) without the use of antibodies. A second approach exploited the mass spectrometric behavior of intact polypeptides. A polypeptide can exist in multiple charge states separated by mass to charge ratio (m/z). Herein, the charge state distribution and the formation of product ions to form selected reaction monitoring (SRM) transitions for intact polypeptide quantitative analysis was evaluated in plasma. Oxyntomodulin, a 37 amino acid anorectic peptide (4449 Da), was employed as a model for analysis in rat plasma. The +7 charge state form of OXM was used to form an SRM for quantitative analysis. Two-dimensional reversed phase ion pair chromatography, a modified solid phase extraction, and a multiply charged SRM of oxyntomodulin enabled a lower limit of quantification of 1 ng/mL. Following development of the LC-MS/MS method, a validation of this approach was performed according to FDA guidelines. Finally, to show further utility of LC-MS/MS, the validated oxyntomodulin method was used in a pharmacokinetic study with sprague-dawley rats. Rats were dosed with oxyntomodulin through intravenous or intratracheal instillation routes of administration. Plasma concentration-time profiles were determined. Using these profiles, noncompartmental parameters were determined for each dose and routes of administration

The Effect of Electronic Cigarette User Modifications and E-liquid Adulteration on the Particle Size Profile of an Aerosolized Product

Electronic cigarettes (e-cigarettes) are an alternate nicotine delivery system that generate a condensation aerosol to be inhaled by the user. The size of the droplets formed in the aerosol can vary and contributes to drug deposition and ultimate bioavailability in the lung. The growing popularity of e-cigarette products has caused an increase in internet sources promoting the use of drugs other than nicotine (DOTNs) in e-cigarettes. The purpose of this study was to determine the effect of various e-cigarette and e-liquid modifications, such as coil resistance, battery voltage, and glycol and drug formulation, on the aerosol particle size. E-liquids containing 12 mg/mL nicotine prepared in glycol compositions of 100% propylene glycol (PG), 100% vegetable glycerin (VG), or 50:50 PG:VG were aerosolized at three voltages and three coil resistances. Methamphetamine and methadone e-liquids were prepared at 60 mg/mL in 50:50 PG:VG and all e-liquids were aerosolized onto a 10 stage Micro-Orifice Uniform Deposit Impactor. Glycol deposition correlated with drug deposition, and the majority of particles centered between 0.172–0.5 μm in diameter, representing pulmonary deposition. The 100% PG e-liquid produced the largest aerosol particles and the 100% VG and 50:50 PG:VG e-liquids produced ultra-fine particles \u3c0.3 μm. The presence of ultrafine particles indicates that drugs can be aerosolized and reach the pulmonary alveolar regions, highlighting a potential for abuse and risk of overdose with DOTNs aerosolized in an e-cigarette system

Interdisciplinary collaboration: A faculty learning community creates a comprehensive LibGuide

Purpose – Many colleges and universities require both undergraduate and graduate students to plan and conduct research as a part of graduation requirements. However, a number of barriers exist for both instructors and students in understanding and conducting research. A small group of, The College at Brockport, instructors who had taught introductory research and research methodology gathered together with librarians as a faculty learning community (FLC) to share information about their instructional methods for teaching research skills. The paper aims to discuss this initiative. Design/methodology/approach – Following an initiative to foster career-span faculty development, The College at Brockport made a three-year commitment to implement a variety of topic-based FLCs beginning in the fall 2008 semester. Findings – Like librarians across the country Brockport librarians have been creating research guides, or “pathfinders,” for decades. The term “pathfinder” was coined in the early 1970s when MIT librarians developed lists of resources and references pertaining to subject disciplines. When LibGuides are marketed, it is not surprising that libraries are quick to adopt this platform to produce pathfinders. LibGuides are chosen because they provide a convenient and simple way to create and update research guides using a live interface, employ web 2.0 technologies in a user-friendly format, and encourage collaboration. Originality/value – Based on the evaluative and qualitative feedback the LibGuide has been refined further. It is a guide that will be under modification as more faculty and students use it

High Sensitivity Mass Spectrometric Quantification of Serum Growth Hormone by Amphiphilic Peptide Conjugation

Amphiphilic peptide conjugation affords a significant increase in sensitivity with protein quantification by electrospray-ionization mass spectrometry. This has been demonstrated here for human growth hormone in serum using N-(3-iodopropyl)-N,N,N-dimethyloctylammonium iodide (IPDOA-iodide) as derivatizing reagent. The signal enhancement achieved in comparison to the method without derivatization enables extension of the applicable concentration range down to the very low concentrations as encountered with clinical glucose suppression tests for patients with acromegaly. The method has been validated using a set of serum samples spiked with known amounts of recombinant 22 kDa growth hormone in the range of 0.48 to 7.65 \mug/L. The coefficient of variation (CV) calculated, based on the deviation of results from the expected concentrations, was 3.5% and the limit of quantification (LoQ) was determined as 0.4 \mug/L. The potential of the method as a tool in clinical practice has been demonstrated with patient samples of about 1 \mug/L

An Analytical Perspective on Determination of Free Base Nicotine in E-Liquids

In electronic cigarette users, nicotine delivery to lungs depends on various factors. One of the important factors is e-liquid nicotine concentration. Nicotine concentration in e-liquids ranges from 0 to \u3e50 mg/mL. Furthermore, nicotine exists in protonated and unprotonated (“free base”) forms. The two forms are believed to affect the nicotine absorption in body. Therefore, in addition to total nicotine concentration, e-liquids should be characterized for their free base nicotine yield. Two approaches are being used for the determination of free base nicotine in e-liquids. The first is applying a dilution to e-liquids followed by two methods: Henderson–Hasselbalch theory application or a Liquid-Liquid Extraction. The second is the without-dilution approach followed by 1H NMR method. Here, we carried out controlled experiments using five e-liquids of different flavors using these two approaches. In the dilution approach, the Henderson–Hasselbalch method was tested using potentiometric titration. The accuracy was found to be \u3e98% for all five e-liquid samples (n = 3). A Liquid-Liquid Extraction was carried out using toluene or hexane as extraction solvent. The Liquid-Liquid Extraction technique was found to be limited by solvent interactions with flavors. Solvent extractions resulted in flavor dependent inaccuracies in free base nicotine determination (5 to 277% of calculated values). The without-dilution approach was carried out using 1H NMR as described by Duell et al. This approach is proposed to offer an independent and alternative scale. None of the methods have established a strong correlation between pre- and postvaporization free base nicotine yield. Here we present comparative results of two approaches using analytical techniques. Such a comparison would be helpful in establishing a standardized method for free base nicotine determination of e-liquids

Acoustic Emissions at an Open Crack

Both finite difference and finite element techniques have been shown to be capable of modeling the propagation of acoustic emissions (AE) [1,2]. However, those calculations could also be done using the simpler Green’s function methods [3]. In this work the finite element method is used to model a problem that includes complexities that cannot be handled using the Green’s function methods