trends in analytical chemistry, vol. 7, no. I,1988 21

Application of electrochemical detection in high-performance liquid chromatography to the assay of biologically active compounds

Toshiharu Nagatsu Nagoya, Japan

and

Kohichi Kojima Hatano, Japan

Electrochemical detection in liquid chromatography was in- troduced in 1973 for the assay of catecholamines, the elec- troactive hormones and neurotransmitterd, and continues to grow in popularity for the assay of trace amounts of bio- logically active compounds in complex samples in biology and medicink*3.

Principles High-performance liquid chromatography with

electrochemical detection (HPLC-ED) is based on electrochemical reactions (oxidation and/or reduc- tion) of electroactive compounds on the electrode surface of an electrochemical detector after separa- tion by HPLC. The general structure of an HPLC-ED instrument is shown in Fig. 1. An HPLC-ED apparatus consists of an HPLC pump, an injector, a column (with or without a temperature controller), and an electrochemical detector (an electrochemical cell, a potentiostat, an I/V convert- er, and a data processor).

Electroactive compounds can be directly mea- sured by ED after separation by HPLC. For exam- ple, dopamine is readily oxidized at an electrode sur- face at low pH to generate the corresponding ortho- quinone, two protons, and two electrons:

The rate at which electrons are transferred across the electrode-solution interface, i.e., the reaction current, is proportional to the concentration of the electroactive species on the electrode surface.

A less electroactive compound can be derivatized to an electroactive compound in three ways: (1) be- fore injection into the HPLC apparatus, (2) by on-

01659936188/$03.00.

line pre-column derivatization, or (3) by on-line post-column derivatization (Fig. 1).

Since gradient elution can cause baseline drift in the detector, isocratic separations are mostly em- ployed in HPLC-ED. The flow should be as pulse- less as possible to minimize baseline noise. HPLC-ED is usually carried out at room tempera- ture, but the stability can be increased by controlling the column and detector temperature. The mobile phase should be deoxygenated to prevent bubbles in the detector cell.

Electrochemical detectors are divided into two categories according to cell design and electrolytic efficiencies, i.e., amperometric and coulometric. Conventional amperometric detectors have a rela- tively small surface area and low electrolytic effi-

Fig. 1. Schematic diagram of an HPLC-ED instrument. (*) A reactor is included, if it is necessary for pre-column or post-col- umn derivatization.

OElsevier Science Publishers B.V.

22

ciency (l-10%), are relatively free from noise in- duced by fluctuations of the liquid flow in the cell, and have a low background current. As a result, al- though the electrolytic efficiency is low, the sensitivi- ty can be high. Coulometric detectors, which have electrodes with a large reactive surface permit the complete reaction of an electroactive compound (100% efficiency), with maximum sensitivity, as compared to conventional amperometric detectors, which react with only a small portion of the com- pound of interest. However, a larger surface area in a conventional thin-layer cell causes a higher back- ground current and noise level, and therefore a de- creased signal-to-noise ratio. Such a cell would also have a large dead volume.

The electrode surfaces are commonly made of glassy carbon, carbon paste, graphite, gold, mercury or platinum. For many bioactive compounds, the most versatile choice is glassy carbon.

Type of electrochemical detectors There are two types of electrochemical detectors:

detectors with one working electrode (single-elec- trode detector) and detectors with two working elec- trodes (dual-electrode detector). The dual-electrode detector can be used in two configurations: parallel adjacent, and in series. The parallel configuration is analogous to a dual-wavelength spectrophotometer, and the peak height ratio at two different oxidation or reduction voltages on the surface of the two elec- trodes provides information on the identity of a peak or estimation of its purity. In the series configura- tion, compounds eluted from the column can be oxi- dized (or reduced) on the first electrode and subse- quently reduced (or oxidized) on the second. Since the electroactive compounds which irreversibly react on the first electrode (for example, tyramine) are eliminated, determination of a reversible elec- troactive compound (for example, dopamine) is pos- sible with high selectivity.

The reduction mode of operation in HPLC-ED is usually difficult, owing to interference by oxygen, and most HPLC-ED assays are carried out using oxidative ED. However, when a compound can be reduced reversibly, a series dual-electrode detector may be used with the first electrode in the reductive and the second in the oxidative mode. In this way in- terference by oxygen is eliminated. Recently micro- bore columns (internal diameter < l-2 mm) have been used to increase the sensitivity of ED. The vol- ume of most commercially available amperometric cells is already quite small and can be decreased to!less than 1 ~1, with no loss of signal, by decreas- ing the spacer thickness. For example, microbore HPLC-ED was applied to the assay of catechol-

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trends in analyticalchemistry, vol. 7, no..l, 1988

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Fig. 2. Typical chromatograms of catecholamines obtained by di- rect injection of lOO_ul of human urine4: (A) anodic response; (B) cathodic response. Peaks: 1 = noradrenaline, 2 = adrenaline, 3 = dopamine, 4 = DOPA. Potentials (vs. AglAgCl): anode, +0.80 V; cathode, +0.20 V.

amines in human urine using a dual-electrode thin- layer cell, with the first and second electrodes in se- ries, set at +0.80 and +0.20 V (vs. Ag/AgCl), re- spectively. The dual-electrode detector enabled se- lective detection of catecholamines among many electroactive species co-existent in urine on the basis of their electrochemical reversibility by monitoring the reduction current at the anode4 (Fig. 2). The de- tection limit for catecholamines using microbore HPLC-ED is 0.05 pg, whereas in conventional HPLC the detection limit is 5 pg.

General considerations for the assay of biologically active compounds

Since HPLC-ED is based on chemical reactions, ED is different from other physical detection methods in HPLC such as UV-VIS or fluorescence detection. Comparison of ED with fluorescence or UV-VIS detection is shown in Table I. ED is more sensitive than UV absorption and equally sensitive to fluorescence detection, with sensitivity as high as radioisotopic methods (minimum detectable quanti- ty, fmol-pmol). The selectivity is determined by the

trends in pnalytical chemistry, vol. 7, no. I, 1988 23

TABLE I. Comparison of electrochemical, fluorescence and spectroscopic detection in HPLC

Detection method Specificity Sensitivity Linear range Stability Maintenance

Electrochemical Selective fmol-pm01 Wide ( 106) Relatively low Relatively easy Fluorescence Very selective fmol-pm01 Medium ( 104-106) Medium Easy uv-VIS Selective for visible absorption pmol-nmol Low (104) High Easy

detection potential. Many bioactive compounds are oxidized at a potential of +800 mV vs. Ag/AgCl. When compounds are detected at lower potentials, the selectivity is high. At higher potentials the noise level rapidly increases, while even more compounds are detected in biological samples than with UV or fluorescence detection. At high sensitivity, without labelling, more compounds can be detected by HPLC-ED than by HPLC-UV or fluorescence de- tection, and this is one advantage of HPLC-ED that makes ED versatile in application. Since many bio- active compounds are electroactive, ED is not very selective, and the retention time in HPLC alone is not enough to identify a compound. Selectivity in HPLC-ED can be increased by using a dual- or multi-electrode detector or three dimensional dis- play (time, potential, and current) of an array cell chromatogram with multi-electrode detectors5 or three-dimensional voltammograms with a rapid scanning voltammetric detector6. HPLC-ED pro- vides a wide linear range, and is very convenient in the assay of bioactive compounds present in biologi- cal samples at various concentrations. For example, we developed an assay procedure for the simulta- neous determination of fifteen monoamines and their metabolites and precursor amino acids from a single tissue sample, and although the concentration of each compound is different in various brain re- gions, it was possible to measure all compounds in samples of brain tissue weighing OS-50 mg’. One drawback of HPLC-ED is its rather low reproduci- bility. In conventional amperometric cells the re- sponse decreases as the electrode surface becomes contaminated. Therefore, recalibration at each as- say, use of a proper internal standard, and regular repolishing of the electrode surface are necessary to get accurate results. A further drawback is that part- ly aqueous solvents are required and gradient elu- tion is difficult to ensure stable detector response. Maintenance in HPLC-ED is relatively easy, espe- cially with a conventional glassy carbon electrode. HPLC-ED is economical since the apparatus and the running cost are generally inexpensive.

Sample preparation in the assay of. bioactive com- pounds should be as simple as possible. Simple de- proteinization of tissue samples and direct injection

of the deproteinized supernatant is preferable. How- ever, in many cases, some interfering substances must be removed prior to HPLC-ED by a sample clean-up procedure such as column chromatography or solvent extraction. Sample clean-up is required to remove substances that interfere with the com- pounds of interest and to remove materials that are adsorbed on the electrode. Sample clean-up is also needed to achieve a long column life. Usually about 1000 samples can be analyzed on one column, but the sample clean-up procedure can prolong the col- umn life and maintain a constant sensitivity of the electrochemical detector.

Assay of biologically active compounds in biology and medicine

Bioactive compounds which can be measured by HPLC-ED with or without derivatization are shown in Table II. Generally speaking, HPLC-ED cannot be applied to the assay of high-molecular-weight compounds, such as proteins and nucleic acids. However, picogram quantities of nucleic acids can be measured by hydrolysis and quantitation of the purine base?.

Even less electroactive compounds can be mea- sured by HPLC-ED by pre-column or post-column derivatization to electroactive species.

One of the best examples of post-column derivati- zation is the assay of acetylcholine and choline using acetylcholine esterase and choline oxidase to con- vert acetylcholine and choline into hydrogen

P erox-

ide which is then electrochemically detected’> ‘. The use of immobilized acetylcholine esterase and cho- line oxidase” makes this assay inexpensive. This method is further improved for the simultaneous de- termination of acetylcholine, choline, noradren- aline, dopamine and serotonin in brain tissues”. The detector system consists of two ED cells aligned in series: a glassy-carbon electrode for catecholamines and serotonin, and a platinum electrode for acetyl- choline and choline. Chromatograms of acetylcho- line, choline, catecholamines, and serotonin are shown in Fig. 3. This method can be successfully ap- plied to the measurement of acetylcholine, choline, noradrenaline, dopamine, and serotonin in the brain of aged rats. Various biologically active amines,

24 trends in analytical chemistry, vol. 7, np. 1,1988

TABLE II. List of biologically active compounds that can be determined by HPLC-ED

Compound type Compound Compound type

Amines and metabolites Catecholamines DOPA

3-0-Methyl-DOPA Tyramine Dopamine Dopamine 3-O-sulfate Dopamine 4-O-sulfate 5-S-Cysteinyldopamine 2-S-Cysteinyldopamine 5-S-Glutathionyldopamine Noradrenaline (norepinephrine) Adrenaline (epinephrine) 3-Methoxytyramine Normetanephrine Metanephrine 3,4-Dihydroxyphenylacetic acid 3,CDihydroxymandelic acid 3,4-Dihydroxyphenylethanol 3,4_Dihydroxyphenylethylene

glycol Homovanillic acid Vanillylmandelic acid 3-Methoxy-4-hydroxyphenyl-

ethylene glycol Salsolinol

Peptides Tyrosine-containing peptides

Disulfide-containing peptides

Porphyrins

Carbohydrates f

Lipids, Steroids Estrogens

Indoleainines 5Hydroxytryptophan Serotonin (5hydroxytryptamine) Melatonin 5-Hydroxyindoleacetic acid Indican

Prostaglandines

Imidazoleamines” Histamine N’-Methylhistamine

Nucleic aciak

Acetylcholineb Acetylcholine Choline

Amino acids and metabolites Vitamins and cofactors Aromatic amino acids

Neurotransmitter amino acidsC

Sulfhydryls (SH)

Disulfides

Tyrosine Thyroxine Thyronine Tryptophan Homogentisic acid

y-Aminobutyric acid (GABA) Glutamic acid Glycine

Cysteine Glutathione Homocysteine

Cystine Glutathione disulfide

Lysined Methionined

a-Keto acids Oxalic acide

Metals

Drugs and chemicals

- Compound

Enkephalines: Met-enkephaline

-Arg -Arg-Phe -Arg-Gly-Leu

Leu-enkephalin -Arg

Bombesin Neurotensin

Vasopressin

Pentacarboxylic porphyrinogens

Monosaccharides Olygosaccharides

Estrone Estradiol Estriol Estetrol 4-Hydroxyestriol

monoglucuronide monosulfate

Catechol estrogens

Leucotriene B, 20-Hydroxyleukotriene B,

Guanine Adenine Uric acid Xanthine Theophylline

Vitamin A Vitamin E

(a-, /I-, y-tocopherols) Vitamin H (biotin) Vitamin C (ascorbic acid) Coenzyme Q

(ubiquinone 9, ubiquinone 10, ubiquinol9, ubiquinoll0)

Copperg

Benzodiazepinesh Carbamazepine Cephalosporins Chlorzoxazone 6-Hydroxychlorzoxazone Cocaineh Cisplatin Digitoxigenin’ Digoxigenin’ Epinine Etoposide (anti-cancer drug) Guanethidine

trends in analytical chemistry, vol. 7, no. I,1988 25

TABLE II (continued)

Compound type

Drugs and chemicals (continued)

Compound

7-Hydroxycoumarin (umbelliferone)

p-Hydroxymethamphetamine p-Hydroxyamphetamine Indalpine (serotonin-uptake

inhibitor) Indoramine (a-adrenergic

antagonist) Isoproterenol Labetalol (a-adrenergic

antagonist) Methylphenidateh Morphine Morphine-6-glucuronide Hydromorphine Normorphine Penicillinsh Penicillamine Phenobarbitalh Phenothiazines

Chlorpromazine Prochlorperazine Thioridazine Trimeprazine

Rauwolfia alkaloids Reserpine Rescinnamine

Sulfonamides

Environmental chemicals Aflatoxins (mutagens, carcinogens, Deoxynivalenol (vomitoxin) toxins, and abuse drugs) 6-Hydroxydopamine

Isoflavons I-Methyl-6phenyl-1,2,3,6-

tetrahydropyridine (MPTP, parkinsonian neuro-

toxin)

Compound type

Environmental chemicals (continued)

Compound

Nitrosamines 11-Nor-d9-tetrahydrocannabinol-

9-carboxylic acid Polycyclic aromatic amines

Enzymes (substrate + product)

Acetylcholine esteraseb (acetylcholine + H,O,)

Choline acetyltransferaseb (choline -+ H,O,)

Tyrosine hydroxylase (tyrosine + DOPA)

Aromatic L-amino acid decarboxylase

(L-DOPA -+ dopamine) (L-5hydroxytryptophan + serotonin)

Dopamine /I-hydroxylase (dopamine + noradrenaline)

Noradrenaline N-methyltrans- ferase

(noradrenaline + adrenaline)

Catechol 0-methyltransferase (noradrenaline + vanillin,

isovanillin) Tryptophan hydroxylase

(L-tryptophan + 5hydroxytryptophan)

Aldehyde dehydrogenase (dihydroxyphenylaceto-

aldehyde --* dihydroxyphenylacetic acid)

y-Glutamyltransferase (y-glutamyl-DOPA -+

DOPA) (glutathione + y-Gl~GlyGly)~

a Pre-column derivatization with sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate or o-phthalaldehyde. b Post-column derivatization to H,O, with acetylcholine esterase plus choline oxidase. ’ Pre-column derivatization with o-phthalaldehyde. d Copper-electrode. e Chemically modified electrode containing surface-bound cobalt phthalocyanate. f Post-column derivatization with copper(I1) bis(phenanthroline) or ethylenediamine. s Pre-column derivatization with dithiocarbamate. h Post-column photolytic derivatization. i Pre-column derivatization with ferrocenoyl azide.

amino acids and peptides, for example, y-aminobu- tyric acid and histamine, can be detected after post- column derivatization with o-phthalaldehyde”. Car- bohydrates can also be measured after post-column derivatization’*.

On-line, post-column, continuous photolytic deri- vatization followed by conventional oxidative ED has been successfully applied to the determination of a number of classes of drugs including phenobarbi- tal, cocaine, pines13.

methylphenidate, and benzodiaze-

Microbore HPLC-ED is ideally suited to the

analysis of electrochemically active neurotransmit- ters (catecholamines and serotonin) and their meta- bolites in dialysate samples in the in vivo brain mi- cro-dialysis method14. A microsampling system for HPLC-ED was constructed for fraction collection, sample preparation, automatic pipetting, auto injec- tion, pre- and post-column derivatization and col- umn switching.

Assay of enzyme activity Chromatographic-photometric measurements of

a substrate or a product in an enzyme reaction have

26 trendsin analyticalchemistry, vol. 7, no. I, 1988

I -1 3 /

-1

-8

-1

1 1

TIME tmfn)

Fig. 3. HPLC elution profiles of an extract from the rat midbrain region in the presence (A) or absence (B) of the immobilized col- umn of acetylcholine esterase and choline oxidas$‘. Peaks: 1 = noradrenaline, 3 = 3,4-dihydroxybenzylamine, 4 = dopamine, 5 = serotonin, 6 = choline, 7 = ethylhomocholine, 8 = acetylcho- line. Adrenaline was not observed.

been used for many years, but the relatively low sen- sitivity of photometric detectors limited the wide ap- plication. HPLC-ED has made the assay of enzyme activity not only rapid but also highly sensitive. HPLC-ED for the assay of enzyme activity is gener- ally based on the rate of appearance of a product, i.e. a discontinuous method, and a sample is withdrawn from the incubation mixture at various times and subjected to HPLC-ED. Since HPLC-ED is highly sensitive, only a small amount of enzyme is required. HPLC-ED assay of enzyme activity can be devel- oped by devising a substrate that produces an elec- troactive product. For example, y-glutamyl- transpeptidase activity can be sensitively assayed by using y-glutamyl-DOPA as the substrate and mea- suring the enzymatically formed DOPA by HPLC-ED”.

In the incubation mixture of an enzyme assay, the concentrations of substrates and cofactors must be optimal. However, in some HPLC-ED methods, the substrate concentration cannot be saturated, be- cause a high substrate peak often interferes with the product peak. In such cases, after incubation the substrate must be removed from the incubation mix- ture by a preliminary purification of the product.

HUMAN PUTAMEN 100 pm01

3 DO

QM- DC

2

QM- DOPA

1

aM- DOPA

OF-

1 I I 1 1 I 1 1 I 1 1 1 I 6420 6 4 2 0 6 4 2 0

TIME (minutes)

Fig. 4. HPLC elution profiles of tyrosine hydroxylase incubation mixture with human putamen homogenate as enzyme source. (I) Blank incubation with b-tyrosine, (2) experimental incubation with t.-tyrosine, (3) blank incubation with o-tyrosine with 100 pmol of L-DOPA added as internal standard. a-Methyl-DOPA (a-M-DOPA, 100 pmol) added to each sample after incubation. Formation of 37.3pmol of DOPA from L-tyrosine during 10 min incubation at 37 “C was calculated from the charts16.

The incubation mixture should be kept as small as possible to get high sensitivity without preliminary concentration of the product. As an enzyme assay by HPLC-ED is highly sensitive but not completely specific, the choice of the blank (control) is impor- tant to ensure the specificity of the assay and to de- tect peaks due to non-enzymatic reaction, contami- nants or other compounds eluted together with the compound. Incubation with a specific enzyme in- hibitor is most desirable for the blank, if a specific in- hibitor is available and it does not interfere with the assay by HPLC-ED. If the enzyme can recognize the stereospecificity of the substrate, incubation with the inactive stereoisomer of the substrate is an ideal blank, as in the case of tyrosine hydroxylase as- say. In the tyrosine hydroxylase assay the product, DOPA, is separated from interfering substances by chromatography on two columns packed with Am- berlite CG-50 and alumina, and is measured by HPLC-ED. As shown in Fig. 4, after the sample clean-up procedure only the peaks of DOPA and the

trends irfanalyticalchemistry, vol. 7, no. I, 1988 27

internal standard, a-methyl-DOPA, are clearly ob- served in the chromatograms. D-Tyrosine is used for the blank incubation16. HPLC conditions should be chosen so as to allow complete separation of the product from the substrate and any interfering sub- stances, and it is preferable to elute the product prior to the substrate, so that the large substrate peak does not interfere with the assay of the product.

3 I. S. Krull, C. M. Selavka, C. Duda and W. Jacobs, J. Liq.

It is possible to monitor the enzyme protein profile of isoenzymes with post-column reactions that pro- duce electroactive products or cofactors. For exam- ple, lactate hydrogenase isoenzymes are separated on an ion-exchange column and their activity is de- termined by post-column detection of NADH pro- duced by the enzymatic oxidation of lactate to pyru- vate*.

Future trends The application of HPLC-ED to the assay of bio-

logically active compounds can be expanded by the use of pre- or post-column derivatization. The sensi- tivity and selectivity of HPLC-ED can be increased from pmol to fmol levels by the use of a microbore column and three-dimensional display with a multi- electrode or a rapid-scanning voltammetric detec- tor. This approach is particularly suited for the anal- ysis of neurotransmitters in perfusate (~1 volumes) obtained by the in viva micro-dialysis technique. Protein profiling can be performed with post-column reactions that produce electroactive products.

4

5

6 7

8

9

10

11

12 13

14

15

16

Chromatogr., 8 (1985) 2845. M. Goto, T. Nakamura and D. Ishii, 1. Chromatogr., 226 (1981) 33. W. R. Matson, P. Langlais, L. Volicer, P. H. Gamache, E. Bird and K. A. Mark, Clin. Chem., 30 (1984) 1477. M. Goto and K. Shimada, Chromatographia, 21(1986) 631. K. Oka, K. Kojima, A. Togari and T. Nagatsu, J. Chroma- togr., 308 (1984) 43. J. B. Kafil, H.-Y. Cheng and T. A. Last, Anal. Chem., 58 (1986) 285. P. E. Potter, J. L. Meek and N. H. Neff, J. Neurochem., 41 (1983) 188. N. Kaneda, M. Asano and T. Nagatsu, J. Chromatogr., 360 (1986) 211. A. F. Spatola and D. E. Benovitz, J. Chromatogr., 327 (1986) 165. N. Watanabe and M. Inoue, Anal. Chem., 55 (1983) 1016. C. M. Selavka, I. S. Krull and K. Bratin, J. Pharmacol. Bio- them. Anal., 4 (1986) 83. A. Carlsson, T. Sharp, T. Zetterstrdm and U. Ungerstedt, J. Chromatogr., 368 (1986) 299. K. Kiuchi, K. Kiuchi, T. Nagatsu, A. Togari and H. Kuma- gai, J. Chromatogr., 357 (1986) 191. T. Nagatsu, K. Oka and T. Kato, J. Chromatogr., 163 (1979) 247.

References 1 P. T. Kissinger, C. Refshauge, R. Dreiling and R. N.

Adams, Anal. Left., 6 (1973) 465. 2 P. T. Kissinger, K. Bratin, G. C. Davis and L. A. Pachla, J.

Chromatogr. sci., 17 (1979) 137.

Toshiharu Nagatsu is Professor at Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan, since 1984. He was Professor of Cell Physiology at the Tokyo Institute of Technology, Yokohama, Japan from 1976 to 1985. He worked at National Institutes of Health, Bethesda, U.S.A. from 1962 to 1964; at the University of Southern California, Los Angeles, U.S.A. from 1967 to 1968; and at the Roche Institute of Molecular Biology, Nutley, U.S.A. from 1972 to 1973. Kohichi Kojima is Chief of the Laboratory of Biochemistry at Hatano Research In- stitute of Food and Drug Safety Center, Hatano, Japan, since 1985. He was a research associate at Tokyo Institute of Technolo- gy, Yokohama, Japan, from 1977 to 198.5, and worked at the Roche Institute of Molecular Biology, Nutley, U.S.A. from 1980 to 1981.

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