Lori Alquier

Method development and validation for the determination of 2,4,6-tribromoanisole, 2,4,6-tribromophenol, 2,4,6-trichloroanisole, and 2,4,6-trichlorophenol in various drug products using stir bar sorptive extraction and gas chromatography–tandem mass spectrometry detection

Recently, haloanisoles and halophenols are associated with multiple product recall situations in the pharmaceutical industry. The majority of the recalls are associated with consumer complaints due to the presence of 2,4,6-tribromoanisole, as extremely low levels of this component can be easily detected by the human nose. As part of the root cause analysis to address the cause of the consumer complaints, a GC-MS/MS based analytical method combined with stir bar sorptive extraction (SBSE) sample preparation was developed for determination of halophenols and haloanisoles from various drug product formulations. The method also applies to the analysis of 2,4,6-tribromoanisole analysis in various packaging materials. The optimized MS/MS method is based on component-specific MRM transitions. The detection limit is component dependent and in the range of 1-100 pg/tablet for solid dosage formulations and 0.04-4 ng/L for water based solutions. Deuterated tribromoanisole was used as internal standard for quantitation. The paper also may provide guidance for performing trace level method validation in the regulated Pharmaceutical Industry.

Stir bar sorptive extraction combined with GC–MS/MS for determination of low level leachable components from implantable medical devices

Identification and determination of leachable components are essential for the safety assessment of implantable medical devices. The safety concern threshold (SCT) for leachable components is 0.15 μg/day for genotoxic or carcinogenic compounds and 1.5 μg/day for others. Regulatory agencies require extraction of a whole medical device using an extraction media that simulates in vitro conditions. Large-sized medical devices therefore require large volumes of aqueous media, leading to extracts of very low concentrations of the targeted analytes. Analysis of these dilute solutions is often challenging, and pre-concentration steps are time consuming and can cause significant sample loss. Stir bar sorptive extraction (SBSE) has proven to be a very useful sample preparation technique that is simple and uses no (or minimal (<1 ml)) aqueous or organic solvents. When combined with a highly selective and sensitive GC-MS/MS analysis, volatile and semi-volatile leachable components can be determined at levels below the SCT of 150 ng/device. An SBSE-GC-MS/MS method using multiple reaction monitoring detection was validated for determination of antioxidant related leachable breakdown products from orthopedic knee-inserts made from ultra high molecular weight polyethylene.

Molecular imaging of drug-eluting coronary stents: method development, optimization and selected applications

A molecular imaging application was developed to characterize the drug distribution on CYPHER® and NEVO™ Drug-eluting Stents using MALDI Qq-ToF analytical methodology. The coating matrix, laser energy, laser frequency, spatial resolution (related to rastering speed) and mass spectrometer parameters were optimized to analyze drug distribution in both durable and biodegradable polymer matrices. The developed method was extended to generate data from stents explanted from porcine coronary arteries. Due to the methods intrinsic specificity, it offers a significant advantage over other techniques in that it allows low-level detection of the target molecule without biological interferences from the blood or tissue. The method is also capable of detecting drug-related degradation products both from the finished stent product and from explanted stents.

Forced degradation studies of rapamycin: Identification of autoxidation products

The immunosuppressant drug rapamycin, also known as Sirolimus, underwent autoxidation under mild conditions to give numerous monomeric and oligomeric compounds, which were generally characterized by size-exclusion chromatography and NP-HPLC with UV and MS detection. Some of the more predominant products, epoxides and ketones, were isolated and identified. Two epoxides and 10S-epimer of rapamycin were described for the first time. Observed rapamycin isomers were also addressed. Computational chemistry was used to provide mechanistic insights. Formation of the majority of the rapamycin products could be rationalized with free radical-mediated autoxidation reactions involving alkene and alcohol sites. Methodological aspects of oxidative stress testing are discussed.

Mass balance in rapamycin autoxidation.

The immunosuppressant drug rapamycin is a complex polyene-containing natural product which undergoes autoxidation. The resulting product mixtures contained numerous monomeric and oligomeric compounds, which represented challenges for addressing mass balance in forced degradation studies and in analysis of aged developmental drug-eluting stents. A combination of SEC with ultraviolet and refractive index detection and RP-HPLC was used to account for drug loss and product formation. The mass balance methodology was subsequently validated for the determination of rapamycin and composite rapamycin autoxidation product material in developmental stent samples. This mass balance approach may find general applicability in other situations where drugs degrade to a plethora of products, which cannot be determined individually and summed.

Evaluation of charged aerosol detector for purity assessment of protein

Commercially available protein reference standard materials are widely used for the quantitation of intact proteins in biopharmaceuticals, food, and consumer products. However, the purity of protein reference standard materials are often assumed to be 100% or they may be assigned an inaccurate value because the methods used to determine protein purity often lack specificity and accuracy. In this study, a high performance liquid chromatography system equipped with a charged aerosol detector (HPLC-CAD) was used for universal response detection to provide a practical, specific, accurate, and robust method for the determination of the purity of protein reference standard materials. This work demonstrates the near uniform CAD responses for six proteins with different molecular weights and different structures. Flow injection analysis (FIA) was used to compare protein responses under various mobile phase and diluent compositions. Similar CAD responses for all six proteins were observed when the mobile phase composition included trifluoroacetic acid (TFA) and acetonitrile. These are typical conditions regularly applied to the separation of proteins by reversed phase (RP) chromatography. The universal response feature of the CAD was employed to determine the purity of Bowman-Birk inhibitor (BBI) reference standard material. This protein is an important ingredient in soybean products and has various therapy applications. Three major components were observed in the commercially available reference standard material by reversed phase gradient HPLC, BBI-Native, BBI-Isoform 1, and BBI-Isoform 2 at relative compositions of 60.0%, 34.2%, and 5.8%, respectively.

Drug-Eluting Stents

Coronary stenting is currently the dominant and most cost-effective interventional approach in managing symptomatic artery diseases. Stent restenosis is the phenomenon of arterial vessel re-narrowing following an angioplasty procedure and is the most common complication of the stenting procedure. Drug-eluting stents (DES) nearly eliminate stent restenosis in simple lesions and significantly reduce its incidence in complex lesions. This chapter provides an overview of the pathophysiology of restenosis and the various considerations that go into the design and development of a successful DES including stent platform and materials, drug carriers, selections of various classes of anti-restenotic agents, and the appropriate modulation of drug load and release duration. Detailed examinations of the leading marketed drug-eluting stents are provided, followed by a brief introduction to devices that are in the development stage which may become the next-generation DES.

Investigation of mass-balance issue in e-beam sterilized paclitaxel eluting coronary stents by SPME/GC-MS.

Paclitaxel eluting coronary stents were sterilized by e-beam in a closed system, to investigate sterilization related mass-balance issues and evaluate potential volatile paclitaxel degradation products. A solid-phase microextraction (SPME) method utilizing a polydimethyl-siloxane/divinyl-benzene (PDMS/DVB) fiber was optimized for extracting the volatiles from the head-space of the sterilized stents. GC-MS was used for separation, identification, and quantitation of the components. Benzaldehyde and benzoic acid were identified as paclitaxel related volatile degradation products. Three groups of stents were included in the study, a control group (not exposed to e-beam), a group sterilized at 25 kGy, and a final group sterilized at 75 kGy. The stents sterilized by e-beam at 75 kGy contained significantly higher levels of benzoic acid relative to the controls and the stents at 25 kGy contained intermediate levels of benzoic acid. The benzaldehyde levels increased in the 25 kGy e-beam sterilized stents relative to the control but remained fairly constant in the 75 kGy e-beam sterilized stents relative to the 25 kGy e-beam results. Mechanism for the formation of benzoic acid and benzaldehyde from paclitaxel was proposed. The levels of benzoic acid and benzaldehyde observed on the stents did not resolve the original mass-balance issue, but most likely contribute to the lack of mass balance observed for paclitaxel.