Ultra-short columns and ballistic gradients: considerations for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis.

Ultra-fast chromatographic separations has enabled fast chromatographic method development and rapid analysis for sample quantification. Decreasing over-all analytical time has become a factor of major importance for all aspects of drug discovery. However, merely decreasing chromatographic analysis time by decreasing k' can lead to inconsistent quantitative or qualitative results due to ineffective separations in complex matrices. We have found that by changing column length and gradient slope we can maintain chromatographic integrity of chemically diverse analytes and achieve the analytical speed required for bioanalytical drug discovery quantitative analysis. We have optimized method development strategy by performing separations on 2x20 mm HPLC columns at flow-rates of 1.5 ml/min to 2 ml/min with full linear gradients achieved in 1 min for the quantification of pharmaceuticals and their metabolites from biological matrices. This method development strategy can be readily adapted to other matrices. This paper will discuss the effects of column length and gradient time in ultra-fast chromatographic resolution.

Ultra-fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods developed for quantification using rapid ('ballistic') gradients on narrow bore, short HPLC columns have been previously described by this laboratory. This paper compares the fast gradient approach with the more traditional high-organic isocratic LC/MS/MS methods. The comparison is based on an analysis of the effectiveness of the chromatographic separations when using the two approaches (i.e. k', N, and W). The data presented herein are derived from actual biological samples analyzed as part of the drug discovery process.

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation.

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).

Application of liquid chromatography-mass spectrometry(n) analyses to the characterization of novel glyburide metabolites formed in vitro.

The application of bench-top ion-trap atmospheric pressure ionization mass spectrometry in the characterization of in vitro metabolites of glyburide is discussed. The metabolites formed in vitro by rat, dog, monkey and human liver microsomes were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (MS)n experiments. The utility of data dependent MS1-MS2-MS3 analyses, where the mass spectrometer makes "real-time" decisions about the experiment to be performed, are described using the characterization of two novel metabolites of glyburide as an example. The metabolite profiles from each species were similar. Six cyclohexyl hydroxylation products were detected, as well as two novel monooxygenation products formed via hydroxylation of the ethyl chain at the benzylic position, and alpha to the amide nitrogen. The ion-trap with electrospray ionization proved to be a sensitive and reliable HPLC detection system that provided important chemical structure information.

Drug quantitation on a benchtop liquid chromatography-tandem mass spectrometry system.

The specificity and selectivity of LC-MS-MS is illustrated to explain why LC-MS-MS has become the method of choice for quantitation within the pharmaceutical industry. Two assays are described that demonstrate the facility with which new ion trap technology can utilize the selectivity and sensitivity of LC-MS-MS to quantitate trace level components within complex matrices, in particular human plasma. One assay undergoes a validation procedure and demonstrates the utility of this new technology for drug quantitation within a regulated environment.

Immobilized human serum albumin: liquid chromatography/mass spectrometry as a method of determining drug-protein binding.

A liquid chromatography/mass spectrometry methodology is described that enables the simultaneous estimation of the individual protein-binding affinities of a mixture of compounds. This approach has proved to be robust and enables a series of molecules to be ranked according to their protein-binding affinities within 20 minutes.

Structural investigations and biological activity of inositol sphingophospholipids from Phytophthora capsici.

Inositol sphingophospholipids that protect pepper (Capsicum annuum c.v. Yolo Wonder) against pathogen have been isolated by chromatographic methods from the mycelium of Phytophthora capsici. The structure of the major compound was determined by chemical methods and mass spectrometry. Phosphodiester bond cleavage of the phospholipid by mild alkaline hydrolysis liberated a ceramide which contained a C16-sphingosine. This long-chain base was identified by gas chromatography and mass spectrometry of its trimethylsilyl derivative. One of the amide-linked fatty acids was found to be 4-hydroxy-2 docosenoic acid. Fast-atom-bombardment mass spectrometry and fast-atom-bombardment collison-induced tandem mass spectrometry were used to characterize the ceramide as N(4-hydroxy-2-docosenoyl)C16-sphingosine. These sphingolipids have a protective effect on cotyledons of young peppers against necrotic lesions induced by the pathogen P. capsici.

Novel fast atom bombardment mass spectrometric procedures for glycoprotein analysis.

We describe procedures, based upon fast atom bombardment mass spectrometry, for characterising the carbohydrate chains in glycoproteins and for determining sites of glycosylation. Strategies for rapidly screening glycoproteins to ascertain the types of sugar chains present and the degree of heterogeneity are presented. Protocols are given for sequencing O- and N-linked glycans. These strategies exploit simple purification steps and afford data at high sensitivity.

Fast atom bombardment mass spectrometric strategies for characterizing carbohydrate-containing biopolymers.

Fast atom bombardment mass spectrometric methodologies for carbohydrate structure determination are intimately linked to classical procedures such as derivatization and hydrolysis. This paper reviews the current status of carbohydrate fast atom bombardment mass spectrometry and reports on two new protocols which we are using to optimize sensitivity and fragmentation.

Location of the O-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan.

The locations of the O-acetyl substituents on the major nonasaccharide repeating unit of the xyloglucan isolated from sycamore extracellular polysaccharides were determined by a combination of analytical methods, including f.a.b.-m.s. and 1H-n.m.r. spectroscopy. The O-2-linked-beta-D-galactosyl residue of the nonasaccharide was found to be the dominant site of O-acetyl substitution. Both mono-O-acetylated and di-O-acetylated beta-D-galactosyl residues were detected. The degree of O-acetylation of the beta-D-galactosyl residue, was estimated by 1H-n.m.r. spectroscopy to be 55-60% at O-6, 15-20% at O-4, and 20-25% at O-3. 1H-n.m.r. spectroscopy also indicated that approximately 50% of the beta-D-galactosyl residues are mono-O-acetylated, 25-30% are di-O-acetylated, and 20% are not acetylated.

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.

GM1 gangliosidosis (type 1) in a cat.

A kitten with clinical and morphological symptoms of a neurovisceral lysosomal-storage disease has been shown to have a marked deficiency of acidic beta-D-galactosidase in the brain, kidney and spleen. Chromatography on concanavalin A-Sepharose and inhibition studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, a selective inhibitor of the neutral broad-specificity beta-D-galactosidase, have shown that the residual beta-D-galactosidase at pH 4.0 in the tissues of the affected cat is due to the neutral beta-D-galactosidase and that there is a complete deficiency of the acidic (lysosomal) beta-D-galactosidase. There is marked accumulation in all tissues and excretion in the urine of neutral oligosaccharides. Analysis of these oligosaccharides by fast-atom-bombardment mass spectrometry and g.l.c. suggests that they arise from the incomplete catabolism of N-glycans of glycoproteins. The ganglioside content of all the tissues is elevated, and it has been shown by t.l.c. that the concentration of a ganglioside fraction with a mobility similar to that of GM1 ganglioside is particularly increased. There is also some evidence of accumulation of glycosaminoglycans in the brain. The clinical symptoms, the complete deficiency of acidic beta-D-galactosidase and the storage products in visceral organs all suggest that this is a case of feline GM1-type gangliosidosis comparable with the severe infantile (Type 1) form of the disease in humans.

A novel mass spectrometric procedure to rapidly determine the position of O-acylated residues in the sequence of naturally occurring oligosaccharides.

A novel procedure is described for the assignment of the position of O-acylated residues in the sequence of oligosaccharides. The procedure is both rapid and sensitive and complements existing carbohydrate techniques. The procedure involves the use of derivatisation, using conditions under which naturally occurring O-acyl groups are stable, and subsequent fast atom bombardment mass spectrometric analysis of the derivatised product. The derivatisation enhances and directs fragmentation providing unambiguous sequence data. The protocol described is shown to be applicable to acetyl groups and to more complex acyl groups such as long chain fatty acids.

Structure of a novel sialylated fucosyl lacto-N-norhexaosylceramide isolated from chronic myelogenous leukemia cells.

A novel sialylated fucosyl glycolipid, which is present at an elevated level in chronic myelogenous leukemia cells, was isolated. The structure of this fucoganglioside was elucidated by methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation, followed by reaction with anti-Lex, Gal beta 1----4 (Fuc alpha 1----3) GlcNAc beta 1----, monoclonal antibody. The structure of this ganglioside was found to be: (Formula: see text). This structure is unique in that a fucose is attached to the internal N-acetylglucosamine but not to the subterminal N-acetylglucosamine. Since this glycolipid is apparently absent in normal granulocytes or acute myelogenous leukemia cells, it can be a specific marker for chronic myelogenous leukemia cells. Based on the structures of this fucoganglioside and normal granulocyte glycolipids, a biosynthetic pathway of extension, sialylation, followed by fucosylation is proposed.

Structural studies of the O-antigen polysaccharides of Klebsiella O5 and Escherichia coli O8.

The O-antigen polysaccharides of Klebsiella serotype O5 and Escherichia coli serotype O8 are serologically very similar or identical. The structures of these two polysaccharides have now been re-investigated. N.m.r. spectroscopy, chromium trioxide oxidation, hydrolysis with a specific phage enzyme, and f.a.b. mass spectrometry were the principal methods used. It is concluded that the O-antigen has the following structure, in which D-Man3Me is 3-O-methyl-D-mannose and n is approximately 10. (Formula: see text) Biosynthetic studies indicate that these antigens are synthesised by addition of D-mannopyranosyl groups to the "non-reducing" end of the mannan chain, and it seems possible that addition of a 3-O-methyl-D-mannopyranosyl group involves termination.

Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells.

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.