Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography-mass spectrometry method for their quantitation.

N-acetyltryptophan (NAT) has long been used as a stabilizer in some protein solutions, such as human serum albumin, to prevent oxidative protein degradation. However, the fate of NAT has not been discussed in literature. Two NAT degradation products have been observed in concentrated albumin solutions (20% and 25%) and identified as 1-acetyl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid and 1-acetyl-3a,8a-dihydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. To monitor the levels of these two previously unidentified NAT degradation products in concentrated albumin solutions, a fully automated method, incorporating online size exclusion chromatography (SEC) trapping and reversed-phase high performance liquid chromatography-mass spectrometry (HPLC-MS) with multiple reaction monitoring (MRM) analysis, has been developed and validated for their quantitative analysis. The method does not require an internal standard. The only sample manipulation is to obtain an albumin concentration of 4% in all standards and test HPLC samples. A limit of quantitation (LOQ) as low as 20 ng/mL has been achieved for both compounds. This method can readily be adopted for the quantitative determination of other small molecules in concentrated protein solutions.

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.

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.

Cytotoxic constituents from the stem bark of Dichapetalum gelonioides collected in the Philippines.

Fractionation of an ethyl acetate-soluble extract of the stem bark of Dichapetalum gelonioides, collected in the Philippines, using the LNCaP (hormone-dependent human prostate) cell line as a monitor, led to the purification of three dichapetalin-type triterpenoids [dichapetalins A (1), I (2), and J (3)], along with two dolabrane norditerpenoids (6, 7), and the additional triterpenoids zeylanol (8), 28-hydroxyzeylanol (9), and betulinic acid. Since compounds 1-3 exhibited promising selectivity against the SW626 (human ovarian cancer) cell line, a re-collection of the plant material was carried out, to obtain more of these compounds for additional biological testing. Two further phenylpyranotriterpenoids [dichapetalins K (4) and L (5)] were isolated from the re-collected plant material. The structures of the new compounds 2-5 and 9 were determined on the basis of spectroscopic data interpretation, and the relative configuration of 6 was confirmed using X-ray crystallography. Compounds 4-6 and the methyl ester, 6a, exhibited broad cytotoxic activity when tested against a panel of human tumor cell lines. Dichapetalin A (1) was not active when evaluated in an in vivo hollow fiber assay in the dose range 1-6 mg/kg.

Strategy for assessing the leachables impact of a material change made in a container/closure system.

Container/closure systems are extensively characterized in terms of their propensity to contribute leachable substances to the drug products they contain. Such a characterization is relevant until a change occurs in the composition or production of the container/closure system itself or the raw materials it is comprised of. When such a change occurs, it is necessary to ascertain the impact that the change would have on the validity and applicability of the previously performed leachables assessment. A general methodology for performing change control evaluations is developed in this manuscript and is illustrated via the use of a case study.

Cytotoxic constituents of Brachistus stramoniifolius.

Phytochemical investigation of Brachistus stramoniifolius (Kunth) Miers (formerly Witheringia stramoniifolia Kunth) (Solanaceae) was initiated following primary biological screening and in view of the absence of prior phytochemical studies conducted on this species. Fractionation of an ethyl acetate extract from the roots of the plant, guided by in vitro cytotoxic activity using cultured KB (human nasopharyngeal carcinoma) cells, led to the isolation of three known compounds of the 13,14-seco-16,24-cyclosteroid type, physalins B (1), F (2) and H (3). Their structures were characterized by comparison of their physical and spectral data to published values, with 1D- and 2D-NMR experiments being performed to assure unambiguous resonance assignments. Biological evaluation of these three compounds against a panel of human and murine cancer cell lines demonstrated their broad cytotoxic activity.

Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of the concentrations of total lycopene and its cis and all-trans isomers in human plasma has been developed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method was based on the observation that, during negative ion atmospheric pressure chemical ionization with collision-induced dissociation, a unique fragment of m/z 467 was formed from the molecular ion of m/z 536 by elimination of a terminal isoprene group. The use of multiple reaction monitoring facilitated the selective detection of lycopene isomers and an internal standard without interference from the isobaric carotenoids a-carotene and beta-carotene, which are also abundant in human plasma. Measurement of total lycopene was carried out using a C18 high-performance liquid chromatography (HPLC) column and an isocratic mobile phase consisting of acetonitrile/methyl tert-butyl ether (95:5) so that all lycopene isomers eluted as a single chromatographic peak. all-trans-Lycopene was separated from its various cis isomers by using a C30 carotenoid column and a gradient solvent system from methanol to methyl tert-butyl ether. The effects of sample preparation and handling parameters on the stability of lycopene were evaluated such as the stability of lycopene in the HPLC autosampler and the effect of saponification upon lycopene isomerization. For example, the half-life of all-trans-lycopene in the HPLC mobile phase in the autosampler at 4 degrees C was determined to be approximately 16 h. Also, saponification of plasma samples was determined to cause lycopene degradation and isomerization so that lycopene recovery was reduced. The accuracy and interassay precision of this LC-MS-MS assay for lycopene showed a standard deviation of less than 10% over the range of 5-500 pmol injected on-column. The limit of detection was 11.2 fmol injected on-column, and the limit of quantitation was 22.8 fmol.