The US regulatory and pharmacopeia response to the global heparin contamination crisis.

The contamination of the widely used lifesaving anticoagulant drug heparin in 2007 has drawn renewed attention to the challenges that are associated with the characterization, quality control and standardization of complex biological medicines from natural sources. Heparin is a linear, highly sulfated polysaccharide consisting of alternating glucosamine and uronic acid monosaccharide residues. Heparin has been used successfully as an injectable antithrombotic medicine since the 1930s, and its isolation from animal sources (primarily porcine intestine) as well as its manufacturing processes have not changed substantially since its introduction. The 2007 heparin contamination crisis resulted in several deaths in the United States and hundreds of adverse reactions worldwide, revealing the vulnerability of a complex global supply chain to sophisticated adulteration. This Perspective discusses how the US Food and Drug Administration (FDA), the United States Pharmacopeial Convention (USP) and international stakeholders collaborated to redefine quality expectations for heparin, thus making an important natural product better controlled and less susceptible to economically motivated adulteration.

Acetonitrile adduct formation as a sensitive means for simple alcohol detection by LC-MS.

Simple alcohols formed protonated acetonitrile adducts containing up to two acetonitrile molecules when analyzed by ESI or APCI in the presence of acetonitrile in the solvent. These acetonitrile adducts underwent dissociation to form a nitrilium ion, also referred to as the substitution ion. Diols and triols behaved differently. In ESI, they formed only one acetonitrile adduct containing one acetonitrile. The S ion was not observed in ESI and was only weakly observed from the dissociation of the (M + ACN + H)(+) ion. On the other hand, the S ion was abundantly formed from the diols in APCI. This formation of acetonitrile adducts and substitution ion from simple alcohols/diols offers an opportunity to detect simple alcohols/diols sensitively by LC-MS interfaced by ESI or APCI. The utility of this chemistry was demonstrated in a method developed for the quantification of cyclohexanol in rat plasma by monitoring the CID-induced fragmentation from the S ion to a fragment ion.

Pertussis toxoid structure: a collaboration and comparison of two-dimensional liquid chromatography-tandem mass spectrometry, ultraperformance liquid chromatography-mass spectrometryE, and capillary liquid chromatography-matrix-assisted laser desorption ionization-tandem mass spectrometry.

The overall structure of pertussis toxoid has been established by analysis of its tryptic digest using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS), capillary liquid chromatography-matrix-assisted laser desorption ionization-tandem mass spectrometry (CapLC-MALDI-MS/MS), and ultraperformance liquid chromatography-mass spectrometry(E) (UPLC-MS(E)). In addition to oxidation and hydrolysis of amino acids losses of terminal peptides are observed. On-line UPLC-MS(E) generated a similar sequence coverage as the other two methods that involved off-line fraction collection. In light of recent favorable comparisons to data-dependent acquisition, UPLC-MS(E) should be the initial method of choice for analysis of a peptide mixture of moderate complexity.

Case study: contamination of heparin with oversulfated chondroitin sulfate.

In late 2007 and early 2008, a cluster of adverse events in patients receiving Heparin Sodium Injection occurred in the United States and in some countries in Europe. The adverse events were reported as being "allergic type" reactions, chiefly characterized by acute hypotension, nausea, and shortness of breath. The root cause of the cluster of adverse events was determined to be a contamination of the heparin by oversulfated chondroitin sulfate. The isolation and structure determination of this contaminant was accomplished by an FDA-led consortium of academic and government laboratories and independently by Baxter Healthcare, whose vial products were first identified in the USA as being associated with the adverse events. Oversulfated chondroitin sulfate was shown to produce acute hypotension in animal models, demonstrating that it was most likely the causative agent responsible for certain of the reported adverse events in patients receiving the contaminated heparin products.

Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection.

Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a high-performance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ~12.5 × 10(-3) nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed.

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS.

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC-MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.

NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12-3.00 ppm.

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00 ppm for heparin API.

Characterization of pertussis toxoid by two-dimensional liquid chromatography-tandem mass spectrometry.

Pertussis toxoid, an acellular pertussis vaccine prepared by hydrogen peroxide treatment in the presence of Fe(3+), has not been well characterized. Because the toxoid has been a part of the DTaP vaccine for infants, it is of interest and significance to have a clear understanding of its structure. The five subunits of pertussis toxin (PT) have a combined molecular weight of approximately 95,000Da. The peroxide treatment in toxoid formation introduces additional complexity into the protein sequence. To maximize sequence coverage, a two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) approach was used to analyze the tryptic digest of toxoid as a whole. An analytical-scale high-performance liquid chromatography (HPLC) instrument using a pentafluorophenyl (PFP) column was used as the first-dimensional LC for fraction collection. The fractions were then analyzed by nanoLC-MS/MS using a C18 column to acquire collision-activated dissociation (CAD) spectra of the tryptic peptides. It is shown that a PFP column has a different peptide retention specificity from a C18 column. A combination of a PFP column and a C18 column is a viable approach for dispersing peptides in a complex mixture. From the structures of 65 peptides that represented approximately 50% of its sequence, PT was found to have sustained heavy oxidative damages during toxoid preparation. Nearly all methionine, cysteine, and (likely) tryptophan residues were oxidized. Evidence of histidine and tyrosine oxidation was also observed. In addition, a large percentage of asparagine was found hydrolyzed to aspartic acid. These findings corrrelate well with the reduction of PT toxicity by peroxide treatment.

Source-induced fragmentation of heparin, heparan, and galactosaminoglycans and application.

Sulfated glycosaminoglycans (GAGs) are difficult molecules for analysis by mass spectrometry due to their complexity in saccharide composition, polydispersity, and sequence heterogeneity. Structural information is typically derived from their enzymatic or chemical digests. Many analytical studies focused on the determination of disaccharide compositions. In this study a direct electrospray mass spectrometry method is described for the analysis of intact heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates. The GAGs were desalted by membrane filtration and analyzed in dilute formic acid (0.5%) in negative ion mode. At the dissociation cone voltage (defined as minimum cone voltage to dissociate all polymeric molecules), heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates produced simple mass spectra consisting primarily of monosaccharide and disaccharide ions derived from glycosidic bond cleavages. The type and abundance of the ions in the dissociation of each molecule were a good reflection of their saccharide compositions. The major ions of heparin were the hexuronic acid ion (m/z 175), glucosamine ion (m/z 240), and the disaccharide ion (m/z 416). Heparan sulfate produced the same set of fragments as heparin since they shared the same compositional saccharides. However, the formation of the m/z 175 ion dominated the source induced fragmentation process for heparan sulfate reflecting its high content of the nonsulfated disaccharide D-glucuronic acid-acetylated glucosamine (GlcA-GlcNAc). Chondroitin/dermatan sulfates contained only acetylated amino sugar (acetylated galactosamine (GalNAc). Consequently, the principle mono/disaccharide ions were all acetylated with m/z values of 282 and 458, respectively. The contrast among the dissociation features of the three types of molecules were sufficient to allow their ready differentiation. Additionally, sensitive detection of chondroitin/dermatan sulfate and heparan sulfate in heparin was made possible by the same differences in the dissociation chemistry of the three types of molecules. As low as 0.5% chondroitin/dermatan sulfate and 5% heparan sulfate in heparin can be reliably detected. This method was successfully used for the detection of oversulfated chondroitin sulfate in heparin as a contaminant following reports of increased adverse events associated with heparin injections from the end of 2007 to early 2008. Heparin is an important, widely prescribed anticoagulant. In light of this contamination event, quality assurance beyond standard activity assays proves to be important. This method provides a simple and fast way for the detection of low level chondroitin, dermatan, and heparan sulfates and their oversulfated derivatives in heparin raw material or formulation.

Characterization of pertussis toxin by LC-MS/MS.

Liquid chromatography-mass spectrometry (LC-MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 degrees C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC-MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.