Publications by authors named "Kati S Hakala"

8 Publications

  • Page 1 of 1

Direct analysis of glucuronides with liquid chromatography-mass spectrometric techniques and methods.

Curr Drug Metab 2010 Sep;11(7):561-82

Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Helsinki, P.O. Box 56, FI-00014 Finland.

Glucuronidation is one of the main phase II metabolic reactions in humans and animals. A variety of analytical techniques and methods have been used for the detection and quantification of glucuronides of both endogenous and xenobiotic compounds from different biological samples of humans and animals. Drug metabolism has been extensively studied with both in vitro and in vivo experiments under various conditions. The purpose of this review is to explore in detail the benefits and drawbacks of different liquid chromatography-mass spectrometric (LC/MS) methods and techniques in detection and identification of all forms of glucuronide conjugates from in vitro, biological, and environmental samples. The entire analytical procedure is covered, from sample treatment, separation, and ionization to qualitative and quantitative analyses. The aim of this review is not to cover every published paper where glucuronides are identified and/or quantified, but rather to focus on special cases where a new analytical approach or technical development has led to a better, more specific, or more comprehensive detection, identification, or quantitation of glucuronide conjugates.
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http://dx.doi.org/10.2174/138920010792927343DOI Listing
September 2010

Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography-ion trap mass spectrometry.

Anal Bioanal Chem 2009 Feb 10;393(4):1327-36. Epub 2008 Dec 10.

Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014, Helsinki, Finland.

Liquid chromatography-ion trap mass spectrometry was used for the detection and structural characterization of metabolites of the anti-obesity drug sibutramine. Metabolites were profiled from incubations of sibutramine in primary cultures of rat hepatocytes. In addition, enantioselectivity of sibutramine metabolism was investigated by carrying out separate incubations with (R)- and (S)-sibutramine. As a result, biotransformation profile for sibutramine with rat hepatocytes is proposed. Nineteen metabolites and several of their isomers formed via demethylation, hydroxylation, dehydrogenation, acetylation, attachment of CO(2), and glucuronidation were identified in MS(2) and MS(3) experiments, though the exact position of the functionality, mostly hydroxylation, could not always be determined from the mass spectrometric information. However, clear enantioselective formation was observed for two hydroxyl derivatives and two glucuronide conjugates, indicating that the hydroxyl/glucuronic acid moiety in those structures is close to the chiral center. Most of the metabolites found in this study are new metabolites of sibutramine, which were not previously reported.
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http://dx.doi.org/10.1007/s00216-008-2540-8DOI Listing
February 2009

Evaluation of cocktail approach to standardise Caco-2 permeability experiments.

Eur J Pharm Biopharm 2006 Nov 30;64(3):379-87. Epub 2006 Jun 30.

Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, Finland.

The purpose of this study was to investigate the suitability and reliability of n-in-one approach using FDA suggested compounds for standardising Caco-2 permeability experiments. Special attention was paid to the evaluation of rank order correlation and mechanistic insights of compound permeability. Transport studies with antipyrine, metoprolol, ketoprofen, verapamil, hydrochlorothiazide, ranitidine, mannitol and fluorescein were performed in 12- and 24-well formats, as single compounds and in cocktails under iso-pH 7.4 and pH-gradient (pH 5.5 vs. 7.4) conditions. Compounds were quantified using n-in-one LC/MS/MS analysis. The cocktail-dosing proved to be a feasible method to determine the permeability of the Caco-2 cell line and to introduce external standards for permeability tests. Even though sink conditions were lost in cocktail experiments for highly permeable compounds, the rank order of compound permeability and the classification to low and high permeability compounds remained unchanged between single and cocktail studies and permeability values of 12- and 24-well formats were directly comparable. Under pH-gradient conditions the margin between high and low permeability compounds was narrower due to the lower permeability (higher fraction of ionisation) of basic molecules. Of the compounds studied, antipyrine, metoprolol, hydrochlorothiazide and mannitol are suitable for evaluation and standardisation purposes of passive permeability, while fluorescein would function as paracellular marker under iso-pH 7.4. As efflux activity may vary between cell batches, verapamil is a useful marker for P-glycoprotein.
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http://dx.doi.org/10.1016/j.ejpb.2006.06.006DOI Listing
November 2006

Metabolite profile of sibutramine in human urine: a liquid chromatography-electrospray ionization mass spectrometric study.

J Mass Spectrom 2006 Sep;41(9):1171-8

Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, CZ 50005 Hradec Kralove, Czech Republic.

We present a detailed experimental approach to detection and subsequent structural characterization of unknown metabolites of sibutramine, using liquid chromatography-mass spectrometric techniques. The full-, precursor ion, and constant neutral loss scan modes of a triple quadrupole mass spectrometer were used for screening sibutramine metabolites in human urine. The structural assessment of unknown metabolites was based on MSn ion trap mass spectrometric analysis and comparison of MSn spectra between the standards and compounds detected. Two phase-I (M1 and M2) and eight phase-II (M3-M6) metabolites of sibutramine were found in human urine. Metabolites M1 and M2, which were found as minor metabolites, originated from N-demethylation of sibutramine. Carbamoyl glucuronides formed from metabolites M1, M2, and their hydroxylated analogs were the main metabolites of sibutramine and were characterized by tandem mass spectrometric analysis and by the chemical modification of their structure. We demonstrate the usefulness of the chemical derivatization approach for estimation of the site of glucuronidation and propose the formation of hydroxylated regioisomers of metabolites M4 and M6.
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http://dx.doi.org/10.1002/jms.1082DOI Listing
September 2006

Feasibility of different mass spectrometric techniques and programs for automated metabolite profiling of tramadol in human urine.

Rapid Commun Mass Spectrom 2006 ;20(14):2081-90

Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, P. O. Box 56, FI-00014 University of Helsinki, Finland.

The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.
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http://dx.doi.org/10.1002/rcm.2562DOI Listing
July 2007

Influence of thaxtomins in different combinations and concentrations on growth of micropropagated potato shoot cultures.

J Agric Food Chem 2006 May;54(9):3372-9

Department of Applied Biology, University of Helsinki, P.O. Box 27, FI-00014, Finland.

Plant-pathogenic Streptomyces species produce a variety of different phytotoxic 4-nitroindol-3-yl-containing 2,5-dioxopiperazines (thaxtomins) that induce scab symptoms on potato tubers (Solanum tuberosum). The possible mutual synergistic or antagonistic effects of thaxtomins are unknown. Modified methodology using column chromatography allowed the purification of thaxtomin A in large quantities (27 mg, HPLC purity of 97%). Thaxtomin A ortho isomer, thaxtomin B, and C-14 deoxythaxtomin B (thaxtomin D) were also purified. All four compounds induced similar symptoms of reduced root and shoot growth, root swelling (10-200 ppb), or necrosis (200-1000 ppb) on micropropagated in vitro cultures of potato. The scab-resistant potato cvs. Sabina and Nicola were more tolerant to thaxtomins than was the scab-susceptible cv. Matilda. Thaxtomins applied in combinations showed additive effects but no synergism, whereas thaxtomins A and B displayed antagonism with thaxtomin A ortho isomer.
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http://dx.doi.org/10.1021/jf060270mDOI Listing
May 2006

Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases.

Anal Biochem 2005 Jun;341(1):105-12

Viikki Drug Discovery Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.
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http://dx.doi.org/10.1016/j.ab.2005.03.003DOI Listing
June 2005

Development of LC/MS/MS methods for cocktail dosed Caco-2 samples using atmospheric pressure photoionization and electrospray ionization.

Anal Chem 2003 Nov;75(21):5969-77

Viikki Drug Discovery Technology Center (DDTC), University of Helsinki, Helsinki, Finland.

Good reliability of Caco-2 permeability studies requires competent sampling and analytical methods to ensure the comparability of day-to-day experiments. In this work, two n-in-one LC/MS/MS methods based on two different ionization techniques were developed and validated for a group of reference compounds; eight of them are recommended by the Food and Drug Administration (FDA) for the evaluation of oral drug permeability. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), as an interface for quantitative LC/MS analysis was evaluated in comparison to the electrospray ionization (ESI). Generally, the validation parameters, including sensitivity, accuracy, and repeatability, were comparable for the APPI and ESI methods. The main difference was that the linear quantitative range of APPI was 3-4 orders of magnitude (r(2) >/= 0.998) whereas in ESI it was typically 2-3 orders of magnitude (r(2) >/= 0.990). By the APPI and ESI methods, the simultaneous analysis of nine highly heterogeneous compounds was achieved within 5.5-7 min, which leads to significant savings in time and cost of the analyses. The successful validation data indicate the usefulness of both the methods for the rapid and sensitive (LOD values typically
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http://dx.doi.org/10.1021/ac034679bDOI Listing
November 2003