Analysis of taxol and related diterpenoids from cell cultures by liquid chromatography—electrospray mass spectrometry

Authentic taxanes (taxol, 10-deacetyltaxol, cephalomannine, 10-deacetylcephalomannine, baccatin III) and extracts from cell cultures derived from various yew tree species have been analyzed by microbore high-performance liquid chromatography (HPLC)—electrospray mass spectrometry (ESMS). All gave excellent positive-ion ES spectra with dominant protonated molecules at low nozzle-to-skimmer bias value (45 V). By increasing the voltage value to 85 V, fragmentation increased and structurally informative spectra were obtained. The fragments found were both of the C-13 side-chain and of the taxane ring, so their analysis gave important information about the taxane structure and any chemical modifications at different positions of the molecule. When tandem MS was used (argon gas, 25 eV collision energy), fragments similar to those obtained from collision-induced dissociation in the source were detected. The cell culture extracts were analyzed by microbore HPLC—ESMS and excellent spectra were obtained on 5–10 ng of separated compounds; even greater selectivity and sensitivity were obtained through use of selected-ion monitoring (SIM). With SIM, 100 pg of all taxanes could readily be detected. In the HPLC—ESMS mode, only 10% of the eluent was mass-analyzed, so 90% would be available for recovery through fraction collecting.     

Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography—mass spectrometry

Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography—mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.     

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.     

Simple liquid chromatography-electrospray ionization mass spectrometry method for the routine determination of salmon calcitonin in serum

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the quantification of salmon calcitonin (sCT) in serum. Serum samples from rats and dogs were deproteinized and freeze-dried. The residue was then reconstituted with 57% acetonitrile in water containing 0.1% trifluoroacetic acid and 0.005% benzalkonium chloride. A 20-microl aliquot of the reconstituted solution was injected onto a polymer based RP-C18 column, The outlet was connected to an ion-trap mass spectrometer equipped with an ESI source, and spectra were recorded in a positive-ion, selected-ion monitoring mode. The limit of quantification of the method was 10 ng/ml. Biexponential curves were observed for the temporal serum concentration of sCT following intravenous administration of sCT to rats (100 microg/kg) and dogs (250 microg/kg), resulting in reasonable pharmacokinetic parameters. The present method appears applicable to routine analysis of serum sCT in pharmacokinetic studies with good selectivity, accuracy and precision.     

Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography–electrospray ionization mass spectrometry

A sensitive LC–MS quantitation method of cetrorelix, a novel gonadotropin releasing hormone (GnRH) antagonist, was developed. Plasma and urine samples to which brominated cetrorelix was added as an internal standard (I.S.) were purified by solid-phase extraction with C₈ cartridges. The chromatographic separation was achieved on a C₁₈ reversed-phase column using acetonitrile–water–trifluoroacetic acid (35:65:0.1, v/v/v) as mobile phase. The mass spectrometric analysis was performed by electrospray ionization mode with negative ion detection, and the adduct ions of cetrorelix and I.S. with trifluoroacetic acid were monitored in extremely high mass region of m/z 1543 and 1700, respectively. The lower limit of quantitation was 1.00 ng per 1 ml of plasma and 2.09 ng per 2 ml of urine, and the present method was applied to the analysis of pharmacokinetics of cetrorelix in human during phase 1 clinical trial.     

Analysis of cyclic and acyclic analogs of retinol, retinoic acid, and retinal by laser desorption ionization-, matrix-assisted laser desorption ionization-mass spectrometry, and UV/Vis spectroscopy.

Laser desorption ionization (LDI)- and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (LDI-MS, MALDI-MS) at 337-nm laser wavelength were used to analyze retinol (vitamin A), retinoic acid, and retinal and their analogs 3-hydroxyretinol, 3-hydroxyretinoic acid, 3-hydroxyretinal, 4-oxoretinol, 4-oxoretinoic acid, 4-oxoretinal, 3,4-didehydroretinol (vitamin A(2)), 3,4-didehydroretinoic acid, 3,4-didehydroretinal, acycloretinol, acycloretinoic acid, and acycloretinal. The compounds exhibit sufficient ionizability which allows to obtain mass spectra by LDI which are similar in quality to those obtained by MALDI. Mass spectra were recorded with a linear time-of-flight (TOF) instrument or a reflectron-type (RETOF) instrument in positive-ion mode. Under the conditions of LDI-MS the compounds form abundant radical molecular ions (M+*), whereas in the MALDI mass spectra abundant protonated molecular ions ([M + H]+) are observed. Characteristic fragment ions provide additional structural information. High-performance liquid chromatography (HPLC) coupled with UV/Vis photodiode detection was used to assist in retinoid characterization. Synthesis of 3-hydroxyretinal, 4-oxoretinal, and acycloretinal was performed by oxidative cleavage of the all-trans-carotenoids of zeaxanthin, canthaxanthin, and lycopene.     

Determination of a cysteine protease inhibitor and its ethyl ester in mouse serum and muscle by liquid chromatography—mass spectrometry

A liquid chromatographic—mass spectrometric method is described for the determination of a cysteine protease inhibitor (E64C) and its ethyl ester in mouse serum and muscle samples. The compounds in the sample, after deproteinization and solid-phase extraction, were separated by isocratic reversed-phase high-performance liquid chromatography and detected by on-line mass spectrometry. The use of an aqueous mobile phase containing methanol and 30 mM ammonium trichloroacetate provided abundant protonated molecular ions of the compounds in the atmospheric pressure chemical ionization interface of the detection system. The method permitted the quantitative determination of the inhibitors without internal standards in the biological matrices. The detections limits for the compounds, in the selected-ion monitoring mode, were 10–15 pmol on-column, at a signal-to-noise ratio of 5.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry

Medium-chain acyl-coenzyme A (CoA) esters are key metabolites in lipid metabolism. Liquid chromatography-electrospray ionization mass spectrometry analysis of medium-chain acyl-CoA esters is described. Eight medium-chain acyl-CoA esters were well separated on a C(8)-MS reversed-phase column using a linear gradient of ammonium acetate buffer (pH 5.3)-acetonitrile. The positive-ion mass spectra of all the saturated and unsaturated medium-chain acyl-CoA esters gave dominant [M+H](+) ions, whereas their negative-ion mass spectra showed abundant [M-H](-) and [M-2H](2-) ions. The positive-ion mode of operation was slightly less sensitive than the negative-ion detection mode. Five medium-chain acyl-CoA esters of C(6:0), C(8:0), C(8:1), C(10:0), and C(10:1) in liver, heart, kidney, and brain from the mouse were identified. The predominant acyl-CoA peaks were C(6:0), C(8:0), and C(10:0). Small amounts of medium-chain acyl-CoAs of C(8:1) and C(10:1) were detected only in heart and kidney. The analytical method is very useful for the analysis of medium-chain acyl-CoA esters in the tissues.     

Mass spectrometry of high-mannose oligosaccharides after trifluoroacetolysis and periodate oxidation.

High-mannose oligosaccharides were treated with a mixture of trifluoroacetic acid and trifluoroacetic anhydride under conditions where reducing terminal N-acetylglucosamine residues are specifically degraded. The resulting mannose-containing products were, after further chemical modifications, analyzed by mass spectrometry in fast atom bombardment and electron ionization modes. Binding positions between monosaccharide residues were deduced from mass spectra of peracetylated compounds, which, prior to the derivatization, had been subjected to periodate oxidation and borodeuteride reduction.     

Characterization of monoglucuronides of vitamin D2 and 25-hydroxyvitamin D2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

The characterization of vitamin D₂ 3-glucuronide, 25-hydroxyvitamin D₂ 3-glucuronide and 25-hydroxyvitamin D₂ 25-glucuronide, biliary metabolites obtained from rats dosed with vitamin D₂ and 25-hydroxyvitamin D₂ per os, was carried out using HPLC-atmospheric pressure chemical ionization (APCI)-MS. The glucuronide obtained from bile specimens was identified by comparison of its chromatographic behaviour with an authentic sample using HPLC—APCI-MS operating in the negative-ion mode. Methylation of the respective fraction with diazomethane gave the methyl ester, which was also confirmed by HPLC—APCI-MS operating in the positive-ion mode. The (M-M)⁻ and (M+NH₄)⁺ ions were monitored in the selected-ion monitoring mode.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

Identification of methotrexate and folic acid analogs by mass spectrometry

Methotrexate and folic acid analogs are polar molecules and attempts to obtain electron impact and chemical ionization mass spectra of these compounds by several laboratories have failed. We have found that methylation of this important class of compounds with diazomethane produces derivatives which are sufficiently volatile to allow their mass spectra to be recorded. The mass spectra of the methyl derivatives of five compounds, 4-amino-4-deoxy-N10-methylpteroylglutamic acid, 4-amimo-4-deoxypteroylglutamic acid, pteroylglutamic acid, 4-amino-4-deoxypteroic acid and N10-methylpteroylglutamic acid are presented, and the fragmentation pathways of these compounds discussed.     

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography—mass spectrometry

A rapid and reliable gas chromatographic—mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether—n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.     

Characteristics of mass spectrometric analyses coupled to gas chromatography and liquid chromatography for 22-oxacalcitriol, a vitamin D3 analog, and related compounds

The characteristics of the mass spectra of vitamin D₃ related compounds were investigated by GC–MS and LC–MS using 22-oxacalcitriol (OCT), an analog of 1,25-dihydroxyvitamin D₃, and related compounds. Fragmentation during GC–MS (electron impact ionization) of TMS-derivatives of OCT and the postulated metabolites gave useful structural information concerning the vitamin D₃-skeleton and its side-chain, especially with respect to the oxidation positions of metabolites. In contrast, few fragment ions were observed in LC–MS (atmospheric pressure chemical ionization), showing that LC–MS gave poor structural information, except for molecular mass. However, when comparing the signal-to-noise ratio (S/N) observed during GC–MS and LC–MS analysis for OCT in plasma extracts, the S/N in LC–MS was over ten-times greater than in GC–MS, possibly due to the low recovery on derivatization and thermal-isomerization in GC–MS. Furthermore, both the GC–MS and the LC–MS allowed the analysis of many postulated metabolites in a single injection without any prior isolation of target metabolites from biological fluids by LC. These results suggest that GC–MS and LC–MS analysis for vitamin D₃ related compounds such as OCT each have unique and distinct advantages. Therefore, the complementary use of both techniques enables the rapid and detailed characterization of vitamin D₃ related compounds.     

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]⁻ together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]⁺), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.     

Extension of the in-gel release method for structural analysis of neutral and sialylated N-linked glycans to the analysis of sulfated glycans: application to the glycans from bovine thyroid-stimulating hormone

This paper reports an extension of the in-gel technique for releasing N-linked glycans from glycoproteins for analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry reported by B. Küster, S. F. Wheeler, A. P. Hunter, R. A. Dwek, and D. J. Harvey (1997, Anal. Biochem. 250, 82-101) to allow it to be used for sulfated glycans. The method was used to identify N-linked glycans from bovine thyroid-stimulating hormone. Following glycan release, either in gel or in solution, the glycans were fractionated directly with a porous graphatized carbon column. The sulfated glycans were examined by MALDI mass spectrometry in negative ion mode with d-arabinosazone as the matrix and both neutral and acidic glycans were examined in positive ion mode from 2,5-dihydroxybenzoic acid. Negative ion post-source decay spectra were also obtained. Twenty-two neutral and fifteen sulfated N-linked glycans were identified and it was shown that negligible loss of sulfate groups occurred even though these groups are often readily lost during MALDI analysis. The glycans were mainly sulfated hybrid and biantennary complex structures. Negative ion post-source decay and positive ion collision-induced fragmentation spectra were obtained.     

Comparison of IR- and UV-matrix-assisted laser desorption/ionization mass spectrometry of oligodeoxynucleotides.

UV-matrix assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) with 3-hydroxypicolinic acid as matrix and IR-MALDI-MS with succinic acid as matrix have proved their feasibility for highly accurate and sensitive mass determination of nucleic acids (DNA and RNA). In this work, a detailed comparison of these two MALDI-methods and between positive- and negative ion mass spectra for the analysis of oligodeoxynucleotides is undertaken. Mass spectra of DNA sequences with up to 40 nucleotides are shown. Both linear and reflectron time-of-flight mass analyzers were used within this study and are compared for their potential in the MALDI analysis of oligodeoxynucleotides. The role of molecule-ion fragmentation is also discussed.     

Comparison of a preliminary procedure for the general unknown screening of drugs and toxic compounds using a quadrupole-linear ion-trap mass spectrometer with a liquid chromatography-mass spectrometry reference technique

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) might be a complement to GC-MS and HPLC-diode array detection for the general unknown screening (GUS) of drugs and toxic compounds, particularly when using information- or data-dependent acquisition (IDA or DDA), an auto-adaptive MS-MS product-ion scan mode where, at each unit time, the m/z ratios above a given intensity threshold are selected for fragmentation. A new quadrupole-linear ion-trap mass spectrometer (LC-QqQlinear ion-trap) was evaluated for GUS using IDA. For the first detection step (so-called "survey scan") the single quadrupole "enhanced" MS mode (EMS), where ions are accumulated then filtered in the Q3-linear ion-trap, was used. The so-called "enhanced" parent ion scan mode (EPI) used at two alternated fragmentation energies gave the best signal intensity and the best mass spectral information when adding mass spectra obtained in low and high fragmentation conditions, respectively, both in the positive (+20 and +50 eV) and negative (-15 and -40 eV) modes. Solid-phase extracts of serum spiked with eight test compounds (chosen for their retention times distributed along the 30-min long chromatogram and for ionising in both the positive and negative modes) were analysed in parallel with this LC-MS-MS technique and with a reference LC-MS method run on a single-quadrupole instrument where low and high in-source fragmentation conditions in the positive and the negative ion modes are alternated. A C(18), 5 microm (150 x 1 mm I.D.) column and a gradient elution of acetonitrile in pH 3, 2 mM ammonium formate, were used for both. Higher signal-to-noise ratios were obtained with the LC-QqQlinear ion-trap instrument than with the reference technique, resulting in mass spectra devoid of contaminant ions and at least as informative as the reconstructed single-MS spectra. After optimisation of the IDA intensity threshold for the detection of tiny chromatographic peaks in noise, five out of the eight compounds (milrinone, lorazepam, fluometuron, piretanide and warfarin) could be unambiguously identified at the concentration of 0.1 mg/l in serum, in the positive or negative modes, or in both, versus only two by LC-MS. All of them could be identified at 1 mg/l by both techniques. These preliminary results show that the sensitivity and mass structural information brought by this new LC-QqQlinear ion-trap instrument may help design an efficient toxicological GUS procedure.