Steven E. Klohr

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structures of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy-and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and delta 1, 10 a-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnostic tool for new degradation products.

Predictive strategy for the rapid structure elucidation of drug degradants

Structural information on drug degradants and impurities can serve to accelerate the drug discovery and development cycle. Traditional structure elucidation methodologies for obtaining this information are often slow and resource-consuming; therefore, LC/MS profiling and LC/MS/MS substructural analysis methodologies have been developed to rapidly and accurately elucidate structures of impurities and degradants. This work is a further development of methodologies used for the elucidation of degradation products of paclitaxel [K.J. Volk et al., Proc. 9th AAPS Ann. Meeting, 1994, p.29]. In this study cefadroxil was used as a model compound for the evaluation of a predictive strategy for the production and elucidation of impurities and degradants induced by acid, base, and heat, using LC/MS and LC/MS/MS profiling methodology, resulting in an LC/MS degradant database which includes information on molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. Furthermore, libraries such as this can provide a predictive foundation for pre-clinical development work involving drug stability, synthesis, and monitoring.

DEVELOPMENT OF REVERSED-PHASE CHIRAL HPLC METHODS USING MASS SPECTROMETRY COMPATIBLE MOBILE PHASES

The majority of chiral HPLC separations are performed in the normal-phase mode using alcohol-modified hexane mobile phases. Normal-phase chiral HPLC methods are not routinely coupled with electrospray ionization mass spectrometry (ESI-MS) because of the mobile phase incompatibility. In this study, we investigated the use of ESI-MS compatible mobile phases for chiral HPLC methods. This would enable the sensitivity and selectivity of LC/MS to be applied to chiral HPLC analyses. We used a commercially available reversed-phase chiral HPLC column (Chiralcelr® OD-R) that permits the use of aqueous organic-modified mobile phases. This paper describes the development of direct, isocratic, and simple reversed-phase chiral HPLC methods for the separation of enantiomers of benzoin, indapamide, 2-phenylbutyric acid, 3-phenylbutyric acid, trans-2-phenylcyclopropane-1- carboxylic acid, verapamil hydrochloride, and pindolol. In addition, we also demonstrate that the reversed-phase chiral HPLC methods developed in this study can be directly coupled with ESI-MS without any modifications. Examples of reversed-phase chiral high performance liquid chromatography-mass spectrometry (RP Chiral-LC/MS) methods are shown for indapamide and pindolol.

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis.

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.

Structural characterization of glycoprotein digests by microcolumn liquid chromatography-ionspray tandem mass spectrometry

An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography.

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

The Coordinated Use of Tandem Mass Spectrometry and High Resolution Mass Spectrometry for the Structure Identification of the Kedarcidin Chromophore

Structure identification strategies for kedarcidin chromophore are described using tandem mass spectrometry (MS/MS) and high-resolution MS techniques. The strategy involves dividing a large, complex natural product molecule into several smaller molecules (substructures) to simplify identification. Substructures are generated using the full-scan MS and MS/MS product ion scan modes. The relationship between substructures, or mass spectral connectivity, is subsequently established using the MS/MS precursor scan mode. Similar to the assembly of a picture puzzle, the structural borders are defined using accurate mass measurements providing molecular formulas of the various substructure molecules. An iterative application of this substructure analysis scheme provides greater substructural detail facilitating identification.

Feasibility of Using Fluorescence Spectrophotometry to Develop a Sensitive Dye Immersion Method for Container Closure Integrity Testing of Prefilled Syringes

A feasibility study was conducted for a sensitive and robust dye immersion method for the measurement of container closure integrity of unopened prefilled syringes using fluorescence spectrophotometry as the detection method. A Varian Cary Eclipse spectrofluorometer was used with a custom-made sample holder to position the intact syringe in the sample compartment for fluorescence measurements. Methylene blue solution was initially evaluated as the fluorophore in a syringe with excitation at 607 nm and emission at 682 nm, which generated a limit of detection of 0.05 μg/mL. Further studies were conducted using rhodamine 123, a dye with stronger fluorescence. Using 480 nm excitation and 525 nm emission, the dye in the syringe could be easily detected at levels as low as 0.001 μg/mL. The relative standard deviation for 10 measurements of a sample of 0.005 μg/mL (with repositioning of the syringe after each measurement) was less than 1.1%. A number of operational parameters were optimized, including the photomultiplier tube voltage, excitation, and emission slit widths. The specificity of the testing was challenged by using marketed drug products and a protein sample, which showed no interference to the rhodamine detection. Results obtained from this study demonstrated that using rhodamine 123 for container closure integrity testing with in-situ (in-syringe) fluorescence measurements significantly enhanced the sensitivity and robustness of the testing and effectively overcame limitations of the traditional methylene blue method with visual or UV-visible absorption detection. LAY ABSTRACT: Ensuring container closure integrity of injectable pharmaceutical products is necessary to maintain quality throughout the shelf life of a sterile drug product. Container closure integrity testing has routinely been used to evaluate closure integrity during product development and production line qualification of prefilled syringes, vials, and devices. However, container closure integrity testing has recently gained industry attention due to increased regulatory agency scrutiny regarding the analytical rigor of container closure integrity testing methods and expectations to use container closure integrity testing in lieu of sterility tests in stability programs. Methylene blue dye is often used for dye ingress testing of container closure integrity, but we found it unsuitable for reliable detection of small breaches in prefilled syringes of drug product. This work describes the suitability and advantages of using a fluorescent dye and spectroscopic detection for a robust, sensitive, and quality control–friendly container closure integrity testing method for prefilled syringes.

Vacuum Decay Container Closure Integrity Leak Test Method Development and Validation for a Lyophilized Product-Package System

A leak test performed according to ASTM F2338-09 Standard Test Method for Nondestructive Detection of Leaks in Packages by Vacuum Decay Method was developed and validated for container-closure integrity verification of a lyophilized product in a parenteral vial package system. This nondestructive leak test method is intended for use in manufacturing as an in-process package integrity check, and for testing product stored on stability in lieu of sterility tests. Method development and optimization challenge studies incorporated artificially defective packages representing a range of glass vial wall and sealing surface defects, as well as various elastomeric stopper defects. Method validation required 3 days of random-order replicate testing of a test sample population of negative-control, no-defect packages and positive-control, with-defect packages. Positive-control packages were prepared using vials each with a single hole laser-drilled through the glass vial wall. Hole creation and hole size certification was performed by Lenox Laser. Validation study results successfully demonstrated the vacuum decay leak test methods ability to accurately and reliably detect those packages with laser-drilled holes greater than or equal to approximately 5 μm in nominal diameter. All development and validation studies were performed at Whitehouse Analytical Laboratories in Whitehouse, NJ, under the direction of consultant Dana Guazzo of RxPax, LLC, using a VeriPac 455 Micro Leak Test System by Packaging Technologies & Inspection (Tuckahoe, NY). Bristol Myers Squibb (New Brunswick, NJ) fully subsidized all work. LAY ABSTRACT: A leak test performed according to ASTM F2338-09 Standard Test Method for Nondestructive Detection of Leaks in Packages by Vacuum Decay Method was developed and validated to detect defects in stoppered vial packages containing lyophilized product for injection. This nondestructive leak test method is intended for use in manufacturing as an in-process package integrity check, and for testing product stored on stability in lieu of sterility tests. Test method validation study results proved the method capable of detecting holes laser-drilled through the glass vial wall greater than or equal to 5 μm in nominal diameter. Total test time is less than 1 min per package. All method development and validation studies were performed at Whitehouse Analytical Laboratories in Whitehouse, NJ, under the direction of consultant Dana Guazzo of RxPax, LLC, using a VeriPac 455 Micro Leak Test System by Packaging Technologies & Inspection (Tuckahoe, NY). Bristol Myers Squibb (New Brunswick, NJ) fully subsidized all work.