ABSTRACT
Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.
Subject(s)
Apoptosis/drug effects , Fruit/chemistry , Neuroblastoma/pathology , Oxidative Stress/drug effects , Plant Extracts/pharmacology , Tumor Suppressor Protein p53/metabolism , Blotting, Western , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Damage/drug effects , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Gene Expression Profiling , Humans , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Protein Transport/drug effects , Tumor Cells, CulturedABSTRACT
Mass spectrometry (MS) is a powerful tool for identification and quantitation of organic molecules from various matrices, especially when combined with liquid chromatography (LC). The aim of this review is to present different MS techniques and methods which can be utilized in drug and metabolism studies using cells and tissues. The first part focuses on the use of LC/MS in permeability studies across cell lines as well as in ABC transporter protein experiments. The second part describes the utilization of MS in drug metabolism studies using cell lines. The third part presents a relatively new application area of MS, namely mass spectrometric imaging (MSI) or imaging mass spectrometry (IMS). Several different MSI techniques can be used for characterization of surfaces, in terms of abundance of proteins and peptides but also small molecules, such as drug compounds and their metabolites, at the surface. The final part gives a review of MS based techniques for direct analysis of cell contents.
Subject(s)
Mass Spectrometry , Pharmaceutical Preparations/metabolism , Animals , Biotransformation , Cell Line , Chromatography, Liquid , Humans , Mass Spectrometry/methods , Permeability , Subcellular FractionsABSTRACT
UNLABELLED: Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 µg/ml of TQ and 72% at 100 µg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha -1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-010-0052-4) contains supplementary material, which is available to authorized users.
ABSTRACT
We have examined the glucuronidation of psilocin, a hallucinogenic indole alkaloid, by the 19 recombinant human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B. The glucuronidation of 4-hydroxyindole, a related indole that lacks the N,N-dimethylaminoethyl side chain, was studied as well. UGT1A10 exhibited the highest psilocin glucuronidation activity, whereas the activities of UGTs 1A9, 1A8, 1A7, and 1A6 were significantly lower. On the other hand, UGT1A6 was by far the most active enzyme mediating 4-hydroxyindole glucuronidation, whereas the activities of UGTs 1A7-1A10 toward 4-hydroxyindole resembled their respective psilocin glucuronidation rates. Psilocin glucuronidation by UGT1A10 followed Michaelis-Menten kinetics in which psilocin is a low-affinity high-turnover substrate (K(m) = 3.8 mM; V(max) = 2.5 nmol/min/mg). The kinetics of psilocin glucuronidation by UGT1A9 was more complex and may be best described by biphasic kinetics with both intermediate (K(m1) = 1.0 mM) and very low affinity components. The glucuronidation of 4-hydroxyindole by UGT1A6 exhibited higher affinity (K(m) = 178 microM) and strong substrate inhibition. Experiments with human liver and intestinal microsomes (HLM and HIM, respectively) revealed similar psilocin glucuronidation activity in both samples, but a much higher 4-hydroxyindole glucuronidation rate was found in HLM versus HIM. The expression levels of UGTs 1A6-1A10 in different tissues were studied by quantitative real-time-PCR, and the results, together with the activity assays findings, suggest that whereas psilocin may be subjected to extensive glucuronidation by UGT1A10 in the small intestine, UGT1A9 is likely the main contributor to its glucuronidation once it has been absorbed into the circulation.
Subject(s)
Glucuronides/biosynthesis , Glucuronosyltransferase/metabolism , Hallucinogens/metabolism , Indoles/metabolism , Psilocybin/analogs & derivatives , Glucuronides/analysis , Glucuronides/chemistry , Glucuronosyltransferase/genetics , Hallucinogens/chemistry , Hallucinogens/isolation & purification , Humans , Isoenzymes/metabolism , Kinetics , Liver/enzymology , Liver/metabolism , Metabolic Detoxication, Phase II , Microsomes/metabolism , Organ Specificity , Psilocybin/chemistry , Psilocybin/isolation & purification , Psilocybin/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Sulfhydryl Reagents/chemistry , UDP-Glucuronosyltransferase 1A9ABSTRACT
The paracellular space defines the passive permeation of hydrophilic compounds in epithelia. The goal of this study was to characterise the paracellular permeation pathway in the human intestinal wall and differentiated epithelial cell models (MDCKII, Caco-2 and 2/4/A1). The permeabilities of hydrophilic polyethylene glycols (PEG) were investigated in diffusion chambers, and mass spectrometry was used to obtain accurate concentrations for each PEG molecule. The paracellular porosity and the size of the pores in the membranes were estimated from the PEG permeability data using an effusion-based approach. The porosities were found to be low (fraction 10(-7)-10(-5) of the epithelial surface) in all investigated membranes. Two different pore sizes (radii 5-6 and >10 A) were detected in the human intestinal epithelium and the Caco-2 and MDCKII cells, while only one (about 15 A) in the 2/4/A1 monolayer. The paracellular porosities of the human small intestine and 2/4/A1 monolayers were larger (>10(-7)) than that of the MDCKII and Caco-2 cells (<10(-7)). We report for the first time the quantitative values describing both porosity and pore size of the paracellular space in the human intestine. The cell models deviate from the small intestine either with respect to porosity (Caco-2, MDCKII) or pore size distribution (2/4/A1).
Subject(s)
Cell Membrane Permeability , Intestinal Mucosa/metabolism , Polyethylene Glycols/pharmacokinetics , Animals , Caco-2 Cells , Cell Line , Dogs , Humans , Intestinal Mucosa/cytology , Jejunum/metabolism , Polyethylene Glycols/chemistryABSTRACT
INTRODUCTION: Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo. METHODS: Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies. RESULTS: Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin. DISCUSSION: Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.
Subject(s)
Celiac Disease/therapy , Gliadin/pharmacokinetics , Peptide Hydrolases/metabolism , Adolescent , Adult , Aged , Biopsy , Caco-2 Cells , Celiac Disease/enzymology , Celiac Disease/pathology , Cell Membrane Permeability/drug effects , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Female , Humans , Inactivation, Metabolic , Intestine, Small/immunology , Intestine, Small/metabolism , Intestine, Small/pathology , Male , Middle Aged , Prognosis , T-Lymphocytes/immunology , Triticum/enzymology , Young AdultABSTRACT
Ionized acetates were used as model compounds to describe gas-phase behavior of oxygen containing compounds with respect to their formation of dimers in ion mobility spectrometry (IMS). The ions were created using corona discharge at atmospheric pressure and separated in a drift tube before analysis of the ions by mass spectrometry. At the ambient operational temperature and pressure used in our instrument, all acetates studied formed dimers. Using a homolog series of n-alkyl-acetates, we found that the collision cross section of a dimer was smaller than that of a monomer with the same reduced mass. Our experiments also showed that the reduced mobility of acetate dimers with different functional groups increased in the order n-alkyl Subject(s)
Acetates/chemistry
, Protons
, Spectrometry, Mass, Electrospray Ionization/methods
, Dimerization
, Hydrogen Bonding
, Ions
, Spectrometry, Mass, Electrospray Ionization/instrumentation
, Structure-Activity Relationship
ABSTRACT
Acyl-CoA-binding protein (ACBP) acts as an acyl-CoA pool former, transporter, and regulator of gene transcription in vitro. We created a transgenic rat line overexpressing ACBP, as the physiological relevance of ACBP in lipid metabolism is unclear. Transgenic rats revealed increased levels of ACBP and significantly elevated acyl-CoA tissue levels while there was no effect on plasma triglyceride, cholesterol, or serum-free fatty acid levels. Metabolic regulators like peroxisome proliferator-activated receptors (PPARgamma, PPARdelta) and sterol regulatory element-binding protein-1 (SREBP-1) messenger RNA levels were significantly reduced (by 23-82%) in liver and adipose tissue of fed transgenic rats, whereas adenosine monophosphate-activated protein kinase (AMPK) protein levels were increased (by 60%). Fasting abolished PPAR downregulation in liver and caused an upregulation in adipose tissue. Administration of AMPK inhibitor reversed SREBP-1 but did not affect PPAR regulation. In conclusion, ACBP acts as an acyl-CoA pool former in transgenic rats and regulates lipid metabolism via SREBP-1 and PPAR regulation. Reduction of SREBP-1 is mediated via increased AMPK levels, whereas regulation of PPARs seems to be mediated by an AMPK-independent mechanism. ACBP itself is a target gene for both transcription factors demonstrating important feedback loops.
Subject(s)
Adipose Tissue/metabolism , Diazepam Binding Inhibitor/metabolism , Liver/metabolism , PPAR delta/metabolism , PPAR gamma/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , AMP-Activated Protein Kinase Kinases , Animals , Animals, Genetically Modified , Cholesterol/blood , Fatty Acids, Nonesterified/metabolism , Lipid Metabolism/physiology , Male , Protein Kinases/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Triglycerides/bloodABSTRACT
This present work describes the systematic experimental comparison of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) for pharmacokinetic (PK) analysis of two drug candidates from rat plasma using single reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The electrospray assay is an established method using a fast liquid chromatography (LC) separation of the sample extracts prior to mass spectrometry analysis. The novel MALDI assays measured the concentration levels of the drug candidates directly from the spotted sample extracts. Importantly, for both LC-ESI and MALDI the same solid-phase sample extraction protocol, internal standards, triple quadrupole mass analyzer platform, and SRM conditions were used, thus effectively standardizing all experimental parameters of the two assays. Initially, analytical figures of merit such as linearity, limit of quantitation, precision, and accuracy were determined from the calibration curves, indicating very similar performance for both LC-ESI and MALDI. Moreover, the LC-ESI rat plasma concentration time profiles of the drug candidates after orally dosing the animals were accurately reproduced by the MALDI assay, giving virtually identical PK results. The direct MALDI assay, however, was able to generate the data at least 50 x faster than the LC-ESI assay. It is shown in this study that analyzing the entire PK curve for one animal took less than 2 min using MALDI (with five replicate analyses per sample), whereas the corresponding LC-ESI assay required 80 min, however, allowing only two replicate measurements in that time frame.
Subject(s)
Pharmacokinetics , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Rats , Rats, Sprague-DawleyABSTRACT
A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.
Subject(s)
Adrenergic alpha-Antagonists/blood , Aqueous Humor/metabolism , Chromatography, High Pressure Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Sulfonamides/blood , Tandem Mass Spectrometry/methods , Adrenergic alpha-Antagonists/pharmacokinetics , Cataract/blood , Cataract/metabolism , Chromatography, High Pressure Liquid/standards , Humans , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/standards , Sulfonamides/pharmacokinetics , Tamsulosin , Tandem Mass Spectrometry/standardsABSTRACT
PURPOSE: To study iris-related complications during cataract surgery in patients on tamsulosin medication. METHODS: Twenty-one consecutive cataract patients administered tamsulosin and 21 control patients were studied. Characteristics of the iris during surgery were recorded. Pupillary diameters of 16 patients were measured before and after iris dilatation. Tamsulosin concentrations in the aqueous humor and serum were analyzed. In five patients, surgery on the second eye was carried out after a 7- to 28-day pause in tamsulosin medication. RESULTS: Each patient administered tamsulosin had a sluggish hypotonic iris, along with a tendency toward miosis and a tendency for prolapse of the iris into the phaco tunnel or into the side port during cataract surgery. Sluggish irises also often adhered to the phaco tip or to the irrigation-aspiration tip. Despite a pause of 7 to 28 days in the use of tamsulosin, the adverse effects persisted. Tamsulosin concentrations varied between 0.1 and 1.0 ng/mL in the anterior chamber fluid. In three of five cases, tamsulosin remained in detectable amounts the aqueous humor after the 7- to 28-day pause. Preoperative pupillary diameter was smaller in the patients using tamsulosin than in the controls. CONCLUSIONS: Tamsulosin has selective alpha1A-adrenoreceptor antagonistic properties and obviously binds for a long period to the postsynaptic nerve endings of the iris dilator muscle, thus affecting iris dilatation and leading to complications in cataract surgery. The iris remained floppy after 7- to 28-day interruption of the tamsulosin regimen.
Subject(s)
Adrenergic alpha-1 Receptor Antagonists , Antineoplastic Agents/adverse effects , Cataract Extraction , Iris Diseases/chemically induced , Iris/drug effects , Sulfonamides/adverse effects , Adrenergic alpha-Antagonists/adverse effects , Adrenergic alpha-Antagonists/pharmacokinetics , Aged , Aged, 80 and over , Antineoplastic Agents/pharmacokinetics , Aqueous Humor/metabolism , Humans , Male , Middle Aged , Miosis/chemically induced , Prolapse , Prostatic Hyperplasia/drug therapy , Pupil/drug effects , Sulfonamides/pharmacokinetics , TamsulosinABSTRACT
A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.
Subject(s)
Prostaglandins/analysis , Animals , Chromatography, High Pressure Liquid , Deuterium , Guinea Pigs , Male , Reference Standards , Spinal Cord/chemistry , Tandem Mass SpectrometryABSTRACT
The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an 'All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of 'clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment.
Subject(s)
Mass Spectrometry/methods , Verapamil/analysis , Verapamil/metabolism , Animals , Chromatography, Liquid , Microsomes, Liver/metabolism , Molecular Structure , Rats , Verapamil/chemistryABSTRACT
Discovery stage studies that address issues of absorption, distribution, metabolism and excretion (ADME) are vital for lead optimization resulting in new drug candidates. Often pharmacokinetics (PK) is assessed in these experiments without regard for the metabolism of the compound or the potential for metabolites to circulate in vivo. This work presents a strategy for drug level determination and detection of metabolites using dried blood spots for sample collection. Initially, metabolites are detected from microsomal incubations and characterized using tandem mass spectrometry. Data dependent enhanced MS and enhanced product ion (EMS-EPI) scanning with dynamic background subtraction was used on a hybrid quadruple linear ion trap mass spectrometer. On-the-fly background subtraction greatly improved the detection of metabolites. These data were used to build a multiple reaction monitoring (MRM) method for the parent and metabolites. MRM-EPI scanning was used to analyze the extracted dried blood spots from the PK study. Circulating metabolites were detected using MRM and their identities confirmed on the basis of fragment ion spectra collected simultaneously. The use of dried blood spots provides a means for re-analysis of PK samples for metabolite identification without the need for complex sample storage and preparation. Both parent compound and metabolite information can be collected in these studies, resulting in a savings of time and resources.
Subject(s)
Blood Specimen Collection/methods , Enzyme Inhibitors/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cathepsin K , Cathepsins/antagonists & inhibitors , Enzyme Inhibitors/blood , Enzyme Inhibitors/pharmacokinetics , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M+H-H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values.
Subject(s)
Cholesterol/analysis , Chromatography, Liquid/methods , Mass Spectrometry/methods , Sitosterols/analysis , Caco-2 Cells , Cholesterol/isolation & purification , Humans , Reproducibility of Results , Sitosterols/isolation & purification , Sterols/metabolismABSTRACT
Trimeric nucleotide building blocks are valuable in synthesis of randomized oligonucleotides. In this study, we have developed HPLC-MS and HPLC-MS/MS methods for quality control of protected trinucleotides. C18 reversed-phase HPLC was used for purity evaluation, and base sequences were verified using negative ion electrospray ionization mass spectrometry (ESI-MS) and collision induced dissociation (CID) MS/MS. The principal dissociation pathway was formation of w-ions, which represent the 3'-5' direction. The other major fragments were d-ions, which are formed by cleavage of 5' C-O bonds of the sugars. The developed method was suitable for verification of purity and structure of the 29 trinucleotides studied.