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Methods of Enzymatic Analysis

Third Edition Editor-in-Chief: Hans Ulrich Bergmeyer Editors: Jürgen Bergmeyer and Marianne Graßl

Volume I

Volume I I

Volume I I I

Volume IV

Volume V

Volume VI

Volume VI I

Volume VII I

Volume IX

Volume X

Volume X I

Volume X I I

Fundamentals

Samples, Reagents, Assessment of Results

Enzymes 1: Oxidoreductases, Transferases

Enzymes 2: Esterases, Glycosidases, Lyases, Ligases

Enzymes 3: Peptidases, Proteinases and Their Inhibitors

Metabolites 1: Carbohydrates Metabolites 2: Tri- and Dicarboxylic Acids, Purines, Pyrimidines and Derivatives, Coenzymes, Inorganic Compounds

Metabolites 3: Lipids, Amino Acids and Related Compounds

Proteins and Peptides

Antigens and Antibodies 1

Antigens and Antibodies 2

Drugs and Pesticides

© V C H Verlagsgesellschaft mbH, D-6940 Weinheim (Federal Republic of Germany), 1986

Distribution:

V C H Verlagsgesellschaft, P.O. Box 1260/1280, D-6940 Weinheim (Federal Republic of Germany)

USA and Canada: V C H Publishers, 303 N.W 12th Avenue, Deerfield Beach F L 33442-1705 (USA)

ISBN 3-527-26052-8 (VCH Verlagsgesellschaft) ISBN 0-89573-2424 (VCH Publishers)

Methods of Enzymatic Analysis

Third Edition

Editor-in-Chief: Hans Ulrich Bergmeyer Editors: Jürgen Bergmeyer and Marianne Graßl

Volume XII Drugs and Pesticides

Editorial Consultant: Michael Oellerich

VCH

Editor-in-Chief: Prof. Dr. rer. nat. Hans Ulrich Bergmeyer Hauptstraße 88 D-8132 Tutzing Federal Republic of Germany

Language Editor: Prof. Donald W. 42 Greenways Esher, Surrey K T 1 0 O Q D United Kingdom

Moss, Ph. D. , D. Sc.

Editors: • Dr. rer. nat. Jürgen Bergmeyer In der Neckarhelle 168 D-6900 Heidelberg Federal Republic of Germany

Dr. rer. nat. Marianne Graßl Frauenchiemsee-Straße 20 D-8000 München 80 Federal Republic of Germany

Editorial Consultant: Prof. Dr. Michael Oellerich Kahlendamm 21 b D-3000 Hannover 51 Federal Republic of Germany

Note The methods published in this book have not been checked experimentally by the editors. Sole responsibility for the accuracy of the contents of the contributions and the literature cited rests with the authors. Readers are therefore requested to direct all enquiries to the authors (addresses are listed on pp. X I X - X X I I I ) .

Previous editions of "Methods of Enzymatic Analysis": 1st Edition 1963, one volume

2nd printing, revised, 1965 3rd printing, 1968 4th printing, 1971

Previous editions of "Methoden der enzymatischen Analyse": 1. Auflage 1962, one volume 2. neubearbeitete und erweiterte Auflage 1970, two volumes 3. neubearbeitete und erweiterte Auflage 1974, two volumes

Library of Congress Card No. 84-105641

2nd Edition 1974, four volumes 2nd printing, 1977 3rd printing, 1981

Deutsche Bibliothek, Cataloguing-in-Publication Data

Methods of enzymatic analysis / Ed.-in-chief: Hans Ulrich Bergmeyer. Eds.: Jürgen Bergmeyer and Marianne Graßl. Weinheim; Deerfield Beach, F L : V C H

Dt. Ausg. u .d .T. : Methoden der enzymatischen Analyse NE: Bergmeyer, Hans Ulrich [Hrsg.] Vol. X I I . Drugs and Pesticides / Ed . consultant: Michael Oellerich. - 3rd edit. - 1986

ISBN 3-527-26052-8 (Weinheim, Basel) ISBN 0-89573-242-4 (Deerfield Beach)

NE: Oellerich, Michael [Bearb.]

© V C H Verlagsgesellschaft mbH, D-6940 Weinheim (Federal Republic of Germany), 1986. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Production Manager: Heidi Lenz Composition: Krebs-Gehlen Druckerei, D-6944 Hemsbach Printing: Hans Rappold Offsetdruck GmbH, D-6720 Speyer Bookbinding: Josef Spinner, D-7583 Ottersweier Printed in the Federal Republic of Germany

Contents

Preface to the Series V Preface to Volume X I I V I I Contents of Volumes I - X I I (Chapter Headings only) X V Contributors X I X

1 Drugs Monitored during Therapy l

1.1 Introduction 2 William E. Evans and Michael Oellerich

1.2 Theophylline 5

Michael Oellerich

1.2.1 Determination with Enzyme-multiplied Immunoassay Technique . . . . 7

1.2.2 Determination with Substrate-labelled Fluorescent Immunoassay 15

1.3 Caffeine 23 Thomas Zysset and Prithipal Singh

1.4 Phenytoin 33 Leoneiio Leone and Emiiio Perucca

1.4.1 Total Phenytoin 37 1.4.1.1 Determination with Enzyme-multiplied Immunoassay Technique . . . . 37 1.4.1.2 Determination with Substrate-labelled Fluorescent Immunoassay 43

1.4.2 Free Phenytoin, Determination by Ultrafiltration and Enzyme-multiplied Immunoassay 50

1.5 Primidone 56

Leoneiio Leone and Emiiio Perucca



1.6 Phenobarbital 69


1.6.1 Determination with Enzyme-multiplied Immunoassay Technique 70


X Contents

1.7 Ethosuximide 82



1.7.2 Determination with Substrate-labelled Fluorescent Immunoassay . . . . 89

1.8 Carbamazepine 95 Thomas M. Li

1.9 Valproic Acid 103 Hans Reinauer

1.10 Digoxin, Digitoxin 112

Gerd Kleinhammer and Hans Mattersberger

1.10.1 Determination with Enzyme-linked Immunosorbent Assay 115


1.11 Methotrexate 134

William E. Evans and Michael L . Christensen


1.11.2 Determination with Enzyme-linked Immunosorbent Assay 144

1.11.3 Determination with Enzyme-inhibition Assay 152

1.12 Amikacin, Gentamicin, Netilmicin, Tobramycin Determination with Enzyme-multiplied Immunoassay Technique . . . . 159 Markus Wenk

1.13 Gentamicin Determination with Competitive Enzyme-linked Immunosorbent Assay 172 Jim Standefer

1.14 KanamycinA 187 Tsunehiro Kitagawa and Kunio Fujiwara

1.15 Viomyein and Ampicillin 200 Tsunehiro Kitagawa

1.16 Disopyramide, Lidocaine, Procainamide, /V-Acetylprocainamide, Quinidine 216 Clarence T. Ueda and Mary C. Haven

Contents X I


1.16.2 Determination with Substrate-labelled Fluorescent Immunoassay . . . . 226

1.17 Nortriptyline 232 Vincent Marks and Graham P. Mould

1.18 Apro tinin 246

1.18.1 Enzyme-linked Immunosorbent Assay 246 Werner Müller-Esterl

1.18.2 Inhibition Assay 257 Marianne Jochum

2 Drugs of Abuse and of Toxicological Relevance 265

2.1 Introduction 266 Christopher P. Price, Peter M. Hammond, R. Stewart Campbell and Tony Atkinson

2.2 Amphetamine (and Methamphetamine) 269 Christopher P. Price and R. Stewart Campbell

2.3 Barbiturates 278 Christopher P. Price and R. Stewart Campbell

2.3 A Determination in Serum with Enzyme-multiplied Immunoassay Technique 280

2.3.2 Determination in Urine with Enzyme-multiplied Immunoassay Technique 284

2.4 Benzodiazepines 291 Christopher P. Price and R. Stewart Campbell

2.4.1 Determination of Diazepam in Serum with Enzyme-multiplied Immunoassay Technique 292

2.4.2 Determination of Oxazepam in Urine with Enzyme-multiplied Immunoassay Technique 297

2.5 Methadone 303 Christopher P. Price and R. Stewart Campbell

2.6 Methaqualone 310 Christopher P. Price and R. Stewart Campbell

X I I Contents

2.7 Morphine and Codeine 317 Christopher P. Price and R. Stewart Campbell

2.8 Benzoylecgonine 325 Christopher P. Price and R. Stewart Campbell

2.9 Phencyclidine 332 Christopher P. Price and R. Stewart Campbell

2.10 Propoxyphene 339 Christopher P. Price andR. Stewart Campbell

2.11 A9-Tetrahydrocannabinol 347 Christopher P. Price andR. Stewart Campbell

2.12 Nortryptiline (and Amitryptiline) 355 Christopher P. Price andR. Stewart Campbell

2.13 Paracetamol (Acetaminophen) 362 Christopher P. Price, R. Stewart Campbell, Peter M. Hammond and Tony Atkinson

2.13 A Determination with Enzyme-multiplied Immunoassay Technique . . . . 364

2.13.2 Determination with Aryl Acylamidase 369

2.14 Salicylic Acid 374 Christopher P. Price, Peter M. Hammond, S. A. Paul Chubb, R. Stewart Campbell and Tony Atkinson

2.15 Nicotine 381 Alberto Castro andNobuo Monji

2.16 Drug Residues in Animal Blood 391 James Fleeker

3 Pesticides 403

3.1 Introduction 404 Hans Ulrich Bergmeyer

3.2 Organophosphorus and Carbamate Residues in Water 406

3.2.1 Radiometric Method 406 Läszlö Horväth

Contents X I I I

3.2.2 Colorimetric Method 412 Gyöngyi Koväcs Huber

3.3 Pesticides in Plants 418 Anna W^dzisz

3.4 Paraoxon in Body Fluids 426 Kenneth W. Hunter, Jr., DavidE. LenzandAlanA. Brimfield

3.5 Atrazine in Water 438 Sigmund J. Huber and Bertold Hock

3.6 Paraquat in Soil 451 Jill P. Benner and Zbigniew Niewola

Appendix 465

1 Symbols, Quantities, Units and Constants 466 2 Abbreviations for Chemical and Biochemical Compounds 469 3 Formulae 478 4 Absorption Coefficients of NAD(P)H 489

Index 491

1.18.2 Aprotinin, Inhibition Assay 257

1.18.2 Inhibition Assay

Marianne Jochum

General

The biomedical use of the low molecular-weight (Mr = 6500) proteinase inhibitor aprotinin (Trasylol®), from bovine tissue cells is based on its broad inhibitory specificity. Major target enzymes are trypsin, chymotrypsin, plasmin, tissue kallikrein and plasma kallikrein. Hence, administration of aprotinin to patients is recommended as part of the therapeutic regimen of various diseases, in particular in shock syndromes, hyperfibrinolysis or acute pancreatitis (for review cf. [1]). Serum or plasma levels of aprotinin obtained after intravenous injection decline rather rapidly due to distribution of the inhibitor in the extracellular fluid and subsequent accumulation, primarily in the kidney [2, 3]. Therefore, one major prerequisite for the optimization of the proteinase inhibitor therapy is the quantitative assessment of the inhibitor levels in patients' plasma or other body fluids.

Application of method: in biochemistry, clinical chemistry and pharmacology.

Substance properties relevant in analysis: aprotinin can be exposed to solutions with extreme pH values [1], Hence, acid treatment of aprotinin-containing plasma samples allows a total recovery and thereby a specific measurement of aprotinin as the only tissue kallikrein inhibitor present in these specimens.

Methods of determination: so far, aprotinin has been measured in biological samples by the trypsin inhibition assay [1 , 4] or by immunological methods [5, 6]. However, these assays are inappropriate for monitoring inhibitor levels in routine clinical diagnosis because of the limited specificity of the enzyme inhibition assay and the prolonged incubation (>40 h) necessary in the immunoassays. The enzymatic determination described here circumvents these difficulties and therefore can conveniently be used as a specific and rapid bedside control of high-dosage proteinase-inhibitor therapy [7].

An enzyme-linked immunosorbent assay was developed only recently ([8], cf. chapter 1.18.1) which provides a versatile means of measuring very precisely and relatively rapidly (4 h) large numbers of aprotinin-containing samples from plasma and other body fluids. This assay is suitable for special purposes, e.g. for monitoring lower dose inhibitor therapy in retrospective studies.

International reference method and standards: not yet available.

258 1 Drugs Monitored during Therapy

Assay

Method Design

Principle

(a) Aprotinin + tissue kallikrein (excess) 5 min

aprotinin-tissue kallikrein + tissue kallikrein (remaining)

(b) D-Val-Leu-Arg-4-NA + H 2 0 tissue kallikrein (remaining)

D-Val-Leu-ArgOH + 4-nitroaniline.

After incubation of acid-treated plasma with tissue kallikrein (EC 3.4.21.35) in excess, the remaining amount of the enzyme is determined by its amidolytic activity on the substrate D-Val-Leu-Arg-4-NA (S-2266). The initial rate at which 4-nitroaniline is released is measured photometrically at 405 nm.

The reaction rate decreases linearly with increasing concentration of aprotinin in the range of 20000 - 80000 K I U * per litre plasma. The concentration of aprotinin is calculated from a calibration curve prepared by diluting normal plasma to which aprotinin (2x 105 KIU/1) has been added before acid treatment.

Optimized conditions for measurement: an enzyme activity of 1 K U * * is used in the assay mixture; taking Km into account, the substrate concentration of 0.15 mmol/1 ensures minimal influence of substrate depletion. Pre-incubation of acid-treated aprotinin-containing samples with the enzyme for 5 min at pH 8.2 is sufficient to achieve complete complex formation. Since the kallikrein activity towards the chromogenic peptide substrate is increased by the addition of plasma, all dilutions of aprotinin-containing plasma samples have to be performed with aprotinin-free plasma instead of buffer: in this way, the same amount of plasma (e.g. 25 ^1) is always added to the assay system.

Temperature conversion factors: the assay should be performed at 37 °C. Conversion factors for other temperatures have not yet been determined.

Equipment

Spectrophotometer or spectral-line photometer capable of exact measurement at 405 nm, provided with a thermostatted cuvette holder; water-bath, centrifuge (Eppendorf 5412); recorder or stopwatch; semi-microcuvettes.

* K I U biological kallikrein inhibitor unit (cf. [1]). ** K U biological kallikrein unit (cf. [1]).


Reagents and Solutions

Purity of reagents: all chemicals should be of the highest analytical grade commercially available.

Preparation of solutions (for about 280 determinations): all solutions in re-purified water (cf. Vol . I I , chapter 2.1.3.2).

1. Buffer (Tris, 0.2 mol/1, pH 8.2):

dissolve 24.2 g tris(hydroxymethyl)aminomethane in 800 ml water, adjust to pH 8.2 with HCl , 1 mol/1 (approximately 100 ml), and dilute to 1000 ml with water (25 °C).

2. Substrate solution (D-Val-Leu-Arg-4-NA, 1.5 mmol/1):

dissolve 25 mg S-2266 (from ABKabiDiagnostics Stockholm, Sweden) in 28.8 ml water.

3. Porcine pancreatic kallikrein solution* (10 6 KU/1):

dissolve the lyophilized enzyme with sterile physiological saline (adjusted to pH 7.5 with triethanolamine) to a stock solution of 106 K U / m l . Prepare further dilutions for the assay (5 x 104 KU/1 ^ 1 KU/20 freshly each day.

4. Normal human plasma:

take blood specimens from veins of at least ten healthy blood donors (9 vol blood and 1 vol sodium citrate solution, 0.1 mol/1). Prepare plasma by centrifugation at 2000 g for 20 min at 4 ° C . Mix equal amounts of plasma from each donor and dispense in small volumes. Treat with perchloric acid (6) as described for samples under Collection and treatment of specimens.

5. Aprotinin standard solutions:

use ampoules with 2 x 105 KIU/10 ml, pH 5, from Bayer AG, Leverkusen; add 10 |al (200 KIU) to 1 ml normal plasma (4) for preparation of the calibration curve. Dilute this stock solution (105 KIU/1) with aprotinin-free normal plasma (4) according to the following scheme:

* Research product from Bayer AG, Wuppertal-Elberfeld.


Stock solution Normal plasma K I U K I U (x 103) (10 5 KIU/1) Hi per assay per 1 untreated

ul plasma

10 190 0.125 10 10 90 0.25 20 20 80 0.50 40 30 70 0.75 60 40 60 1.00 80 50 50 1.25 100

6. Perchloric acid solution (PCA, 30 g/1):

dilute 8.57 ml 70% (w/v) PCA with 191.43 ml water.

7. Potassium carbonate solution ( K 2 C 0 3 , 5 mol/1):

dissolve 6.91 g K 2 C 0 3 in 8 ml water and dilute to 10 ml with water.

Stability of solutions: store all solutions at 2 ° C to 8 ° C . Solutions (1) and (7) are stable for two months i f not contaminated with micro-organisms. Solution (2) is stable for six months i f prepared with sterile water and kept in the dark. Solution (3) is stable for at least two weeks. I f not contaminated, solutions (5) and (6) are stable indefinitely. Normal plasma (4) is stable for at least 3 - 4 months at - 20 °C or more than 6 months at - 7 0 ° C . Commercially available lyophilized normal plasma may also be used.

Procedure

Collection and treatment of specimens: take blood specimens from the vein without stasis (9 vol blood + 1 vol sodium citrate solution, 0.1 mol/1). Prepare plasma by centrifugation at 2000 g for 20 min at 4 ° C .

Acid treatment of plasma samples: incubate plasma (e.g. 0.5 ml) for 10 min at 25 °C with an equal volume of PCA solution (6). Centrifuge for 10 min at 10000 g, neutralize ca. 0.980 ml supernatant with ca. 20 \i\ K 2 C 0 3 solution (7), keep at 4 ° C to 8 °C for further 30 min and thereafter centrifuge again for 10 min. Freeze the resulting supernatant at - 2 0 ° C or below.

Stability of aprotinin in the sample: aprotinin in plasma is stable at room temperature at least for 1 day or at 4 ° C for 2 days. Aprotinin solutions of pH < 4 should be used within a few hours. Neutralized aprotinin-containing plasma samples can be stored for 1 month at - 2 0 ° C or for 6 months at - 7 0 ° C . Repeated freezing and thawing (n = 10) has no influence on the inhibitory activity of aprotinin in plasma.


Details for measurements in other samples: the same procedure may be used for measurements in serum and other specimens.

Assay conditions: wavelength Hg 405 nm; light path 10 mm; final volume 1 ml; temperature 37 °C (thermostatted cuvette holder). Measure against air. Before starting the assay, adjust temperature of solutions to 37 °C.

Establish a calibration curve using aprotinin solution (5) instead of sample.

Measurement

Pipette successively into the cuvette:

blank sample concentration in assay mixture

Tris buffer (1) normal plasma (4) sample or standard (5) kallikrein solution (3)

0.855 ml 0.025 ml

0.020 ml

0.855 ml

0.025 ml 0.020 ml

Tris 171 mmol/1

aprotinin up to 1250KIU/1 kallikrein 1000 KU/1

D-Val-Leu-Arg-4-NA 0.15 mmol/1

mix thoroughly with a plastic spatula, wait for 5 min,

Tris 171 mmol/1



substrate solution (2) 0.100 ml 0.100 ml

Tris 171 mmol/1



mix and read absorbance each min or monitor the reaction on a recorder over a period of 5 min.

Tris 171 mmol/1



I f the amount of aprotinin in the sample exceeds 1.0 K I U per assay, plasma samples must be diluted with aprotinin-free, acid-treated normal plasma (4).

0.125 Ö.25 0.5 0.75 1.0 1.25 KJU/assay F i g * c a l i b r a ' i o n

curve for the assay of apro-10 20 40 60 80 100x103 KIU/I plasma tinin in plasma.


Calibration curve: calculate (&A/At)sampk in percent of (AA/At)blSink. the percent of free enzyme activities of the standards against their activity concentrations of aprotinin (Fig. 1).

Calculation: the inhibitory activity of aprotinin in the sample (patients' plasma) is taken from the calibration curve.

Validation of Method

Precision, accuracy, detection limit and sensitivity: the within-run imprecision for aprotinin, 4 x 104 KIU/1 was 1.8-10.8% in 8 series; the between-series imprecision (n = 8) was 4.4%. The recovery for 2 x 104 to 8 x 104 KIU/1 (working range) was 90 -125%. Data on accuracy are not available since standard reference material is not established yet.

The detection limit is 0.125 K I U per assay, i.e. 104 KIU/1 of untreated plasma. Sensitivity is found to be AA/At = 0.002/min at 405 nm (Eppendorf photometer), the mean 1.23-fold increase caused by pooled plasma. With an individual increase in

Sources of error: since kallikrein activity is increased 1.23-fold (n = 90; RSD = 7.2%) by addition of acid-treated normal plasma, all dilutions of aprotinin-containing plasma samples must be made with aprotinin-free, acid-treated plasma. Plasma samples without acid treatment cannot be used because of a rather high and irregular stimulation of the kallikrein activity in the assay system. The same holds true for serum. Dilution of aprotinin-containing plasma samples with buffer or isotonic saline solution instead of plasma yields too low inhibitor concentrations.

Occassionally, aprotinin-free plasma samples from a single individual may stimulate kallikrein activity to a lesser (up to 1.1-fold) or greater (up to 1.5-fold) extent than kallikrein activity lower than 1.15-fold or higher than 1.3-fold, the calibration curve and dilutions of aprotinin-containing plasma samples should be performed with the individual patient's plasma (pre-aprotinin infusion sample) instead of pooled plasma.

Addition of NaN 3 to aprotinin-containing plasma as a preservative before acid treatment partly destroys the inhibitor activity during the subsequent procedure.

Specificity: under the given conditions the substrate S-2266 is split specifically by the tissue kallikrein used in the assay.

Therapeutic ranges: continuous i.v. infusion of 250000 K l U / h in polytraumatized patients resulted in a mean plasma concentration of 4.5 x 104 KIU/1 [7].


References

[1] H. Fritz, G. Wunderer, Biochemistry and Applications of Aprotinin, the Kallikrein Inhibitor from Bovine Organs, Drug Res. 33 (I), 4, 479-494 (1983).

[2] H. Fritz, K.-H. Oppitz, D. Meckl, B. Kemkes, H. Haendle, H. Schult, E. Werle, Verteilung und Ausscheidung von natürlich vorkommenden und chemisch modifizierten Proteinaseinhibitoren nach intravenöser Injektion bei Ratte, Hund (und Mensch), Hoppe-Seyler's Z . physiol. Chem. 350, 1541 -1550 (1969).

[3] H. Kaller, K. Patzschke, L . A. Wegner, F. A. Horster, Pharmacokinetic Observations Following Intravenous Administration of Radioactive Labelled Aprotinin in Volunteers, Eur. J . Drug Metab. Pharmacokinet. 2, 79-85 (1978).

[4] H. Fritz, I. Trautschold, E. Werle, Protease-Inhibitors, in: H. U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Vol. / / , Verlag Chemie, Weinheim, and Academic Press, New York 1974, pp. 1064-1080.

[5] T. Shikimi, Sandwich Enzyme Immunoassay of Aprotinin, J . Pharmcobio-Dyn. 5, 708-715 (1982). [6] A. Eddeland, K. Ohlsson, A Radioimmunoassay for Measurement of Human Pancreatic Secretory

Trypsin Inhibitor in Different Body Fluids, Hoppe-Seyler's Z. physiol. Chem. 359, 671 - 675 (1978). [7] M. Jochum, V. Jonäkovä, H. Dittmer, H. Fritz, An Enzymatic Assay Convenient for the Control of

Aprotinin Levels during Proteinase Inhibitor Therapy, Fresenius Z. anal. Chem. 317, 718-719 (1984).

[8] W. Müller-Esterl, A. Oettl, E. Truscheit, H. Fritz, Monitoring of Aprotinin Plasma Levels by an Enzyme-Linked Immunosorbent Assay ( E L I S A ) , Fresenius Z. Anal. Chem. 317, 717-718 (1984).

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