Polyamine determination in clinical laboratories by high-performance liquid chromatography

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Polyamine determination in clinical laboratories by high-performance liquid chromatography T. Matsumoto* and T. Tsuda Nagoya,Japan and

0. Suzuki Hamamatsu,Japan The current statusof high-performance liquid chromatography (HPLC) for the determinationof polyamines and their acetylderivativesis presented. Reversed-phase HPLC, with ion-pairing agents dissolved in the mobile phase, is now gaining in popularity because of its high resolving power. The use of microcolumn systemsfor polyamine assaysis giving promising resultsfor the analysisof small samples such as biopsy specimens. A new capillary zone electrophoresis method which has been developedfor polyamine analysisin our laboratoryis also briefly described.

Introduction Polyamines, such as putrescine, spermidine, spermine and their acetylated derivatives, are widely accepted to be closely associated with normal cell and neoplastic growth and have been reported to facilitate transcription, translation, RNA and protein synthesis’>‘. The metabolic pathways for polyamines are briefly summarized in Fig. 1. In 1971, Russe13 reported higher urinary excretion of polyamines in cancer patients; this discovery made polyamines candidates for tumor markers. Since then many reports describing clinical studies of polyamines have appeared. One of the best examples is the proceedings of an international symposium held in Gifu, Japan in 19844 where nearly half the papers presented at the symposium are devoted to clinical studies. Very recently, we have reviewed clinical polyamine studies5 and have drawn the same conclusions as previous authors6 that polyamines can be utilized for both the screening and diagnosis of a cancer according to tumor kind and tumor activity. An especially good correlation is observed between cerebrospinal fluid (CSF) polyamines (particularly putrescine) and malignant brain tumors such as medulloblastoma and glioblastoma, and between poly-

* To whom all correspondence 01659936/90/$03.00.

should be addressed.

amines in urine or blood and acute hematological malignancies. However, many reports have shown that polyamine measurements are not very effective for early diagnosis of cancer. In recent clinical studies of polyamines, therefore, attention has been directed toward longitudinal studies which aim to follow-up the effectiveness of therapies, to predict response to treatment and to identify relapse of cancerslT4. A number of investigators have obtained good results along these lines. Recently, we have found a rapid and drastic rise in putrescine and N’-acetylspermidine in’ tissues subjected to organ failure induced by endotoxin shock7, obstructive jaundice and portal vein ligation. These results and other evidence suggest that putrescine may be a marker of organ failure. For the analysis of polyamines and acetylpolyamines high-performance liquid chromatographic (HPLC) methods are most widely used for research purposes and sometimes for clinical investigation. We present here a review of the current status of their determination by HPLC and include a brief description of their separation by capillary zone electrophoresis, a relatively new method for polyamine analysis. Some new HPLC methods are briefly summarized in Table I. HPLC determinations of polyamines In 1974, Samejima et aZ.*reported an HPLC method for polyamines and since then a number of papers have appeared on the subject. Methods reported in 1976-1980 are summarized in great detail in a review by Fujita et a1.‘. We have counted as many as 30 papers on the HPLC analysis of polyamines published in the last 8 years (1981-1988) in our file. Over that time the methods have been improved to make them more rapid and sensitive, and to give better resolutionlo. Recent efforts have also been directed toward assays for acetylated polyamines and unusual polyamines. Samples with a high protein content are usually deproteinized with trichloroacetic acid or perchloric acid as the initial step, then subjected to a simple clean-up procedure prior to analysis by HPLC. These pretreatment procedures enhance column life and maintain constant sensitivity. The commonest 0

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293 ,

1

L-Ornithioe

1

1 I

I

I

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+I

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Omithins lecarboxylase (OOC)

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1 NWeoacetylsparmine

1

CN~CONH(CH2),NN(CN2)rNN(CH2)~NH2

‘Op$

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Fig. 1. Pathwaysfor biosynthesisand interconversion ofpolyamines in mammalian cells.

method employed is extraction with an organic solvent, especially n-butanol. Various mini-columns or paper have also been employed for rapid isolation of polyamines. Dowex-50, Amberlite CG-50, Bio-Rex 70, Whatman P81 paper, and Sep-Pak silica cartridges have all been used in this way. The classical cation-exchange HPLC columns, such as Aminco PA, Shimadzu ISC-OYSO504 and Partisil-1OSCX are still useful for the separation of polyamines in biological samples. Current commercially available ion-exchange resins permit much higher capacit and higher resolution than previously. N1- and N lY -acetylspermidines can be completely separated with these columns under suitable conditions. In our laboratory, we use a column (5 x 0.4 cm I.D.) of a CK-10 U cation exchanger (Mitsubishi Kasei, Tokyo, Japan), and obtain good results for the separation of 11 free polyamines, acetylpolyamines, and histamine derivatives, as shown in Fig. 2. The details of the experimental conditions are shown in Table II. C.V. values of this method are less than 6.3% for each polyamine. With due care and maintenance, more than 300 samples can be analyzed with one column by this system. Reversed-phase HPLC with columns of C,, C,, or CN silica is now gaining general popularity (Table I). Ion-pairing agents such as octanesulphonic

acidlo~ll, heptanesulphonic acid (see Table I, N. Brown, 1982) and triacetylammonium phosphate12 can also be added to the mobile phase. This ion-pairing technique combines the advantages of reversedphase and ion-exchange chromatography and thus sometimes makes it possible to obtain much better resolution. We have recently introduced ion-pairing reversed-phase HPLC14 to our laboratory to give the results shown in Fig. 3. We use a Shim-pack CLCODS column (15 x 0.6 cm I.D.) (Shimadzu, Kyoto, Japan) for reversed-phase HPLC6. A gradient elution system with methanol-sodium perchlorate buffer in the presence of hexanesulphonate as an ionpairing reagent is programmed with a micro-computer; the details are shown in Table II. The C.V. values of this method are less than 6.9% for each polyamine. Ion-pairing reversed-phase HPLC seems more suitable for the determination of acetyl derivatives of polyamines than ion-exchange HPLC; acetylputrescine does not overlap any of the amino acids appearing in the chromatograms. For detection of amine peaks, fluorimetry is the most popular method for both ion-exchange and reversed-phase HPLC. The post-column eluates are generally derivatized with o-phthalaldehyde for fluorimetric detection; however, pre-column derivatization with dansyl chloride is also reported in many

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294 TABLE

I. Comparison of published methods for HPLC determinations

of polyamines

Abbreviations used: FA = fluorescamine; OPA = o-phthalaldehyde; FMOC = 9-fluorenylmethyl chloroformate; Put = putrescine; Cad = cadaverine; Spd = spermidine; Spm = spermine; A-Put = acetylputrescine; A-Cad = acetylcadaverine; NlA-Spd = N’-acetylspermidine; N8A-Spd = NE-acetylspermidine; NlA-Spm = N’-acetylspermine. Reference

Column (size)

Derivative

Elution

Mobile phase

Compound

Total run time

Sensitivity

Material

K. Samejima, J. Chromatogr., 96 (1974) 250 Anal. Biochem., 76 (19xx) 392

Vydac reversedphase (500x3mm I.D.)

FA

Reversed-phase linear gradient

Methanolsodium borate flow-rate 1 ml&n

Put, Cad, Spd,

20 min

lo-50 pm01

Tissue serum urine

N. A. Seiler, J. Chromatogr., 221(1980) 227

PBondapak Cts (300 X 3.9 mm I.D.)

OPA

Ion-pairing reversed-phase

Sodium acetateacetonitrile flow-rate 1.5 mlhnin

21 compounds of amino acids, polyamines and acetyl polyamines

51 min

50 pm01

Tissue urine

P. K. Bondy, 1. Chromatogr., 224 (1981) 371

Partisil PXS lo-25 SCX (250 x 3.5 mm I.D.)

OPA

Ion-exchange linear gradient

Sodium acetatesodium nitrate flow-rate 0.7 ml/mm

GABA, A-Put, NlA-Spd, N8A-Spd, Put, Spd, Spm, y-glutamyl-Put, 2-hydroxyputrescine

80 min

20 pm01

Tissue urine

M. M. AbdelMonem, J. Chromatogr., 222 (1981) 363

Micropak CN-10 (250 x 2.5 mm I.D.) Ultrasphere-Si (150 x 4.4 mm I.D.)

Dansyl-Cl

Reversed-phase gradient (sequential two step HPLC)

A: n-Hexanemethyleneisopropanol B: Chloroformisopropanol A: flow-rate 3 ml/min B: flow-rate 2 ml/mm

A-Put, NlA-Spd, NIA-Spd 1,6-Diaminohexane

45 min

N. Brown, J. Chromatogr., 245 (1982) 101

PBondapak Cts (300 x 3.9 mm I.D.)

Dansyl-Cl

Ion-pairing reversed-phase

1-Heptanesulphonic acidacetonitrile flow-rate 2 mlhnin

F’w Spd,Spm,

37 min (28 min)

N. A. Seiler, J. Chromatogr., 339 (1985) 45

Ultrasphere-IP (250 x 4.6 mm I.D.)

OPA

Ion-pairing reversed-phase

Sodium acetateacetonitrile flow-rate 1 ml/min

24 Compounds of amino acids, polyamines and acetyl polyamines

95 min

M. A. Desiderio, J. Chromatogr., 419 (1987) 285

LiChrosorb RP-18 (250 x 4 mm I.D.)

Dansyl-Cl

Reversed-phase gradient

Disodium hydrogenphosphate-acetonitrile flow-rate 1.8 ml/min

GABA, Put, Cad 1,7-Diaminoheptane Spd, Spm

32 min

l- 10 pm01

Tissue

P. Koski, Anal. Biochem., 164 (1987) 261

Spherisorb S5 ODS2 (250 x 4.6 mm I.D.)

Dabsyl-Cl

Reversed-phase gradient

Sodium acetateacetonitrile flow-rate 1.5 ml/min

A-Put, A-Cad, A-Spd, A-Spm, Put, Cad, Spd, Spm

22 min + re-equilibrium time

2 pm01

Bacteria

C. Loser, J. Chromatogr., 430 (1988) 249

Nova Pak Cts (150 x 3.9 mm I.D.)

OPA

Ion-pairing reversed-phase

Boric acid-potassium hydrochloride buffer flow-rate 1.5 mhmin

A-Put, A-Cad, NlASpd, N8A-Spd, NlASpm, Put, Cad, Spd

25 or 56 min

0.5-l

Tissue serum urine

T. Y. Chou, J. Chromatogr., 454 (1988) 169

Radial-Pak Resolve Cts (100 x 8 mm I.D.)

FMOC

Isocratic reversed-phase

Acetonitrilewater flow-rate 1.5 ml/min

Put,Cad

14 min

21-24 pmol

Urine

N. Watanabe, Clin. Chem., 35 (1989) 1694

Cis Biophase-III (150 x 6 mm I.D.)

Isocratic ion-pairing reversed-phase

Potassium-phosphate buffer flow-rate 0.4 ml/min

Put, Cad, Spd, Spm

40 min

0.3-4 pm01

Urine

Reversed-phase

Methanol-water flow-rate 1 ml/min

1,3_diaminopropane A-Put, NlA-Spd, N8A-Spd, NlASpm, Put, Spd,

60min

5-25 ng

Urine seminalplasma

Ion-exchange step gradient

Sodium phosphate-NaCl buffer flow-rate 0.7 ml/min

A-Put, A-Cad, NlASpd, NSA-Spd, NlASpm, Put, Cad, Spd, Spm, His, 1-Methylhistamine

70 min

l-10 pm01

Tissue serum urine

S. Wongyai, J. Liq. Bio-Sil ODS-5S (250 x 4 mm Chromatogr., I.D.) 12 (1989) 2249

Benzoilchloride (UV)

Spm

1,6-Diaminohexane

Urine

25 fmol

Red blood cell Urine

pm01

1,7-Diaminoheptane

Spm T. Matsumoto, The Physiology of Polyamines, Vol. II, 1989, p. 219

Mitsubishikasei CK 1OU (50 x 4 mm I.D.)

OPA

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100’

1

15.

SO25

L 10

20

30

40

50 (min)

0

10

20

30

40

50 (min)

Fig. 2. Polyamine analysisby ion-exchange HPLC. The chromatogram on the left shows separation of the authenticpolyamines and their related compounds and on the right that of the urine extractfrom a gall bladder cancer patient. For experimental conditions, see Table I. Key: I = acetylputrescine; 2 = acetylcadaverine; 3 = histamine; 4 = I-methylhistamine; 5 = Ns-acetylspermidine; 6 = N’-acetylspermidine; 7 = putrescine; 8 = cadaverine; 9 = N’-acetylspermine; 10 = spermidine; 11 = spermine. For the reference chromatogram (left, 100 pmoles of each amine except spermine (200pmoles) were injected on-column.

TABLE

II. Our conditions of HPLC for polyamine analyses Ion-exchange HPLC

Ion-pairing reversedphase HPLC

Instrument

Shimadzu LC4A

Shimadzu LC6A

Column

Mitsubishi-Kasei CK-1OU 50 X 4mmI.D.

Shim-pack CLSODS 150 x 6 mm I.D. or Shimadzu Techno Research STR ODSM 150 X 6 mm I.D.

Mobile phase

(A) 0.2 M sodium citrate-O.5 M NaCl (B) 0.2 M sodium citrate- 1.OM NaCl (C) 0.2 M sodium citrate-2.5 M NaCl

(A) 0.1 M sodium perchlorate (pH = 4.0)-10 mM hexanesulphonate (B) 25% (A) buffer (pH = 3.0)-75% methanol

Gradient

Step gradient

Programmed gradient

Mobile phase flow-rate

0.7 ml/min

1.Oml/min

Column reaction temperature

70 “C

50 “C

Reaction reagent

o-Phthalaldehyde

o-Phthalaldehyde

Reaction flow-rate

0.5 ml/min

0.3 ml/min

Detector

Shimadzu RF-535 Ex = 345 nm, Em = 455 nm

Shimadzu RF-535 Ex = 345 mu, Em = 455 nm

IO

20

30

40

50lmin)

0

IO

20

30

40

50 (min)

Fig. 3. Polyamine analysisby ion-pairing reversed-phase HPLC. The chromatogram on the left shows separation of authenticpolyamines and their related compounds and on the right that of a urine extract from a bladder cancer patient. For experimental conditions, see Table I. Key: 1 = L-omithin; 2 = acetylputrescine; 3 = putrescine; 4 = acetylcadaverine; 5 = cadaverine; 6 = histamine; 7 = N’-acetylspermidine; 8 = N’-acetylspermidine; 9 = spermidine; 10 = N’-acetylspermine; 11 = spermine. For the reference chromatogram (left panel), 100 pmol of each amine were injected into the HPLCport.

papers (Table I). Three papers have appeared using pre-column derivatization with o-phthalalde-hyde12’13. The detection limit of recent fluorimetric HPLC systems is usually l-10 pmoles in the injected volume. Fluorescamine can also be used in place of o-phthalaldehyde, but the latter is now more popular because of its lower cost and higher sensitivity. Watanabe et al. l5 reported the electrochemical detection of polyamines with immobilized enzymes in a post-column reactor which is a more specific and more sensitive method. Very recently Cheou and Krull (see Table I, T.Y. Cheou, 1988) reported a new fluorogenic reagent, 9-fluorenylmethyl chloroformate, for free polyamines, which gives high stability of the reaction product but the method awaits assessment by other research groups. Separation by capillary electrophoresis Capillary zone electrophoresis with a high voltage is a relatively new separation method, in which electroosmotic flow carries the solute to the detector16>17.A high voltage (100-1000 V/m) is applied to an open capillary column, 50-200 pm I.D., 30-150 cm in length, to give analysis times of the same order as those by HPLC. Capillary electrophoresis is becoming increasingly popular, and has been widely accepted due to its high separating power; it has been applied to pharmaceutical and medical samples with considerable success. We have separated polyamines by capillary electrophoresis as an alternative to HPLC for clinical use. The concentrations of polyamines in human urine

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1001

,^w:.J kh

/

4

“h, 0

2

4

6

8

10

12

14

i ;LJCd

16

16

20

22

24

26 (min)

1

h/* y.+__JJ L_& f

0

2

4

6

8

10

12

14

16

16

20

22 (min)

Fig. 4. Polyamine analysis by capillary electrophoresis. The left trace shows separation of the authentic freepolyamines and the right one that of rat small intestine extract (a biopsy sample after bile duct ligation). Key: 1 = hexamethylenediamine; 2 = putrescine; 3 = cadaverine; 4 = spermidine; 5 = spermine. For the reference chromatogram (left panel), 0.7pmol of each amine except hexamethylenediamine, was injected in the capillary.

and blood are at @ml levels and it is, therefore, necessary to use a high sensitivity detector if the sample is not concentrated during the pre-treating procedure .

We used the standard pre-treating procedure for excluding proteins, following pre-labelling with fluorescamine on a micro scale. Then the labelled sample was separated by capillary electrophoresis, and detected on-column with a fluorescence detector. A typical example is shown in Fig. 4. The experimental conditions were as follows: column: fused silica capillary tubing, 50 cm x 50 ,um I.D., medium: borate buffer-KCl-NaOH 5 mM (pH 9.0) and, ethylene diamine 0.1%. The amount injected and the minimum absolute amount detectable were lo-15 nl and around 100 fmol, respectively. In capillary electrophoresis, a solute is carried by electroosmosis. In the presence of sodium dodecylsulphate (SDS)-micelles in the medium, the solute also distributes itself between the SDS-micelle and the non-micellar aqueous phase. We must also consider these factors in order to prevent adsorption on the inner wall of the capillary. The instrumentation for capillary electrophoresis is quite simple. A highvoltage supply (20-50 kV, maximum current 200-500 ,BA) is not expensive due to the low current demand. The column is an open capillary tube and the medium is mostly free aqueous solution. In addition to the simple mechanism and instrumentation, capillary electrophoresis requires only very small samples thereby allowing analysis of polyamines in biopsy specimens. Conclusion Modern HPLC techniques permit high resolution and sensitivity. Closely similar analogues such as N1and N*-acetylspermidines can be completely separated by ion-pairing reversed-phase HPLC; detec-

tion limits obtained with fluorimetry are as low as l-10 pmoles. The techniques are mature enough for no great advance to be made in the principle of separation. However, great efforts are being made to improve the HPLC systems in detail. HPLC is not suitable for polyamine analysis of a large number of samples because it generally requires 30-60 min for one run. This problem can be overcome to some extent by automatization of the system (including an autosampler controlled by a microcomputer). The second improvement will be miniaturization of the HPLC column and its system, which will make the sample size required much smaller18>19.Various type of microcolumns or narrow-bore columns and their attached systems are now becoming commercially available and should be applied to polyamines. This miniaturization will be useful especially for accurate determination of very low levels of polyamines or acetylpolyamines in samples such as plasma and CSF, or their assay in very small samples, such as biopsy tissues. Acknowledgement This work was supported by a Grant-in-Aid for Cancer Research from Ministry of Education, (62010037). References 1 U. Bachrack and Y. M. Heimer (Editors), The Physiology of Polyamines, Vols. 1 and 2, CRC Press, Boca Raton, FL, 1989. 2 A. E. Pegg and P. P. McCnn, Am. J. Physiol., 243 (1982) C212. 3 D. H. Russell, Nature New Biol., 233 (1971) 144.

4 K. Imahori, F. Suzuki, 0. Suzuki and U. Bachrach (Editors), Polyamines: Basic and Clinical Aspects, VNJ, Utrecht, 1984. 5 T. Matsumoto and 0. Suzuki, in U. Bachrach and Y. M. Heimer (Editors), The Physiology of Polyamines, CRC Press, Boca Raton, FL, 1989, p. 219.

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6 D. H. Russell and B. G. M. Durie (Editors), Polyamines as 7

8 9 10 11 12 13 14

Biochemical Markers of Normal and Malignant Growth, Raven Press, New York, 1978. T. Matsumoto, M. Tokoro, A. Hori, Y. Nimura, S. Shionoya, T. Takahashi and 0. Suzuki, Proceedings of the Znternational Conference on Polyamines in Life Sciences, 1986, p. 219. K. Samejima, M. Kawase, S. Sakamoto, M. Okada and Y. Endo, Anal. Biochem., 76 (1976) 392. K. Fujita, T. Nagatsu and K. Shinpo, in S. Parvez, T. Nagatsu, I. Nagatsu and H. Nagatsu (Editors), Methods in Biogenic Amine Research, Elsevier, Amsterdam, Ch. 32, p. 741. N. Seiler, J. Chromatogr., 379 (1986) 157. J. Wagner, C. Danzin and P. Marmout, J. Chromatogr., 227 (1982) 349. T. Skaaden and T. Greibrokk, J. Chromatogr., 247 (1982) 111. R.L. Heideman, K.B. Fickling and L.J. Waker, Clin. Chem., 30 (1984) 1243. K. Gamoh and T. Fujita, Shimadzu Hyoron, 44 (1987) 55.

15 N. Watanabe, M. Asano, K. Yamamoto, T. Nagatsu, T. Matsumoto and K. Fujita, Chem. Lett., (1988) 1169. 16 J. W. Jorgenson and D. Lukas,Anal. Chem., 53 (1981) 1298. 17 T. Tsuda, K. Nomura and G. Nakagawa, J. Chromatogr., 264 (1983) 385. 18 T. Ishizuka, K. Ishikawa, M. Maseki, Y. Tomoda and T. Tsuda, J. Chromatogr., 380 (1986) 43. 19 P. Kucera (Editor), Micro Column High-Performance Liquid Chromatography, Elsevier, Amsterdam, 1980.

Takatoshi Matsumoto is Lecturer at the First Department of Surgery, Nagoya University School of Medicine, 65 Tsuruma-cho, Nagoya 466, Japan. Takao Tsuda is Associate Professor at the Department of Applied Chemistry, Nagoya Instituteof Technology, Nagoya 466, Japan. Osamu Suzuki is Associate Professor at the Department of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan.

Fractals and fractal-like concepts in chemical analysis Horatio A. Mottola Stillwater,OK, U.S.A. Fmctul surfaces and fracti-like concepts are finding an increased interest in the physical sciences. This note briejly discusses two aspects of relevance to chemical analysis: (i) fmctul surfaces and the immobilization of analytical reagents, and (ii) fractal-like kinetics in continuous-flowsystems. ‘Once you get over the hump, and you understand the paradigm, you can start actually measuring things and thinking about things in a new way. You see them differently. You have a new vision. It’s not the same as the old vision at all - It’s much broader.’ Christopher Scholz

The word fractal was coined by Mandelbrot in 1975l and comes from the Latin ‘fractus’, meaning rough and broken up. It singles out, in essence, geometrical concepts. As a result of attention to detail, it modifies, and actually upgrades, the concepts of Euclidean geometry. Euclidean geometry and its parameters (length, depth, and thickness) fail to describe irregular shapes. A very large number of physicochemical phenomena encountered in contemporary analytical chemistry take place at irregularly defined boundaries or interphases. Most of our conceptual views of these phenomena, however, are given by Euclidean views of space in three dimen016%9936/90/$03.00,

sions, planes in two dimensions, lines in one dimension, and points in zero dimension. Mandelbrot2, popularizing fractals, went beyond the Euclidean dimensions of 0, 1,2, and 3, and introduced fractional (fractal) dimensions which allow one to attach a number to the irregularity of an object (surface, boundary). The number and character of these irregularities and the duration of processes at them are keys to conceptualization of some fractal situations in chemical analysis. The fractal dimension categorizes boundaries, and kinetics in fractal-like environments (erroneously named as ‘fractal-like kinetics’) characterizes processes taking place at (or in the vicinity of) those type of boundaries. Fractal thinking will continue to spread into the physical sciences but so far it has received only token attention in the subdiscipline of chemistry concerned with chemical identification and measurement (i.e. chemical analysis). Two examples of fractal situations in this subdiscipline are briefly presented here.

Fractal surfaces of analytical interest Chemical reagents in immobilized form are used in a myriad of analytical situations, spreading beyond stationary phases in chromatography. Today’s analytical chemist makes use of them for preconcentration purposes, in enzyme reactors, and in optical and electrochemical sensors, to name only the most 0 Elsevier Science Publishers B .V.