Histamine in Fish and Theanine in Tea Two Cases for Employing Planar Chromatography

Histamine in Fish and Theanine in Tea Two Cases for Employing Planar Chromatography Prof. Dr. Karl Speer S. Kretzschmar, H. Dunger, I. Kölling-Speer Technische Universität Dresden Institut für Lebensmittelchemie Bergstr. 66, D-01062 Dresden 1

Biogenic amines 2

Why fish is a problem High water content and soft protein Enzymes show high activity at low temperatures Some fish species obtain high contents of free amino acids N N H NH 2 Histamine 3

Histamine toxication Short incubation time Decrease of the blood pressure Dizziness Gastric spasms and headache Nausea and vomiting 4

Histamine in food < 50 ppm are safe 50-200 ppm may be toxic 200 500 ppm are toxic 500 1000 ppm are toxic and dangerous 5

Legal regulations in Germany Paragraph 16 FischhygieneVO and guideline 95/71/EU Scombridae, Clupeidae, Engraulidae, Coryphaenidae and Istiophoridae > 200 mg Histamine/kg» Marketing ban Engraulidae in salt brine > 400 mg Histamine/kg» Marketing ban Appendix 3 of Fischhygiene-VO with guideline 91/493/EU 6

Analysis of biogenic amines Extraction Derivatisation Clean-up Separation & Detection 7

Development of a TLC - screening-method CAMAG Automatic TLC Sampler III CAMAG TLC Scanner II 8

Extraction 10 g of the homogenized sample + 80 ml TCA (10%) Ultra-Turrax 90 s adjust to 100 ml with TCA and filter 9

Derivatisation 1 ml of the filtrate is adjusted to ph 8 Add 1 ml borate-puffer and 2 ml dansyl chloride solution Incubate for 1 h Adjust to 10 ml with water SO 2 CL O 2 S N H R + R-NH 2 - HCL N(CH 3 ) 2 N(CH 3 ) 2 10

Clean-up Add 5 ml of diethyl ether Shake vigorously and centrifuge Remove the organic layer and shake the water residue twice more Reduce the combined extracts to dryness and dissolve the residue in 5 ml acetonitrile Separation TLC plates silica gel 60 (Merck) (20 x 10 cm, layer thick-ness 0.25 mm, pre-washed with developing solvent) Separation was performed in a horizontal development chamber 11

Densitogram of a standard solution (365/>400 nm) PHE TYR HIS SPM SPD CAD PUT benzene : chloroform : triethylamine (10:6:7) 12

Densitogram of a spiked fish extract PUT SPD SPM TYR HIS PHE benzene : chloroform : triethylamine (10:6:7) 13

Densitogram of a spiked fish extract SPM/HIS CAD SPD TYR PHE/Matrix benzene : chloroform : triethylamine (10:6:2) 14

Validation data for histamine Linearity 50 250 mg/kg Recovery (200 mg/kg) 108 % Confidence interval (n = 5, P = 0,95) LOD DIN 32645 ± 9.9 mg 17.5 mg/kg LOQ DIN 32645 56 mg/kg 15

Densitograms of two fish samples Matrix Mackerel smoked 178 mg/kg histamine Matrix Tuna in oil 2804 mg/kg histamine 16

Histamine contents using different methods TLC HPLC ELISA Fluorimetry Mackerel, smoked Salmon, smoked 178 174 251 - n.d. <5 <1 1 Tuna in oil 2804 2541 2893 - Tuna (Interlaboratory tests) 39 - - 37 Contents of histamine in mg/kg 17

Theanine charakteristic constituent of tea 50 % of the total free amino acids theanine content in tea leaves c. 0.2-2 % sweeter taste HOOC NH 2 O N H pleasant tranquilising or gentle freshening effect 18

Densitogram of a standard mixture Glutamic acid Theanine chloroform:methanol:glacial acetic acid (75:20:5) 19

Detection Excitation wavelength of dansylated amino acids (Literature data): 250-370 nm; CAMAG: 313 nm Kretzschmar et al.: 365 nm for histamine Peak area at excitation wavelength 365 nm and 313 nm 1600 1200 Detection Area 800 > 400 nm 400 0 Theanine Glutamic acid 20

Sample preparation: 0.2 g of ground tea are extracted with cold water (15 ml) (10 min in an ultrasonic bath) Content [%] Content [%] 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Theanine single extraction twofold extraction 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Theanine Glutamic acid Glutamic acid cold water extraction hot extraction 21

Densitogram: black tea, Nepal Theanine Glutamic acid 22

Validation data Theanine Glutamic acid Linearity 0.045 3.75 % Recovery 94 % 99 % Confidence interval (n = 5, P = 0,95) ± 0.02 % ± 0.01 % LOD* 0.10 % 0.08 % LOQ* 0.35 % 0.28 % * DIN 32645 23

Contents of theanine and glutamic acid in different teas 35 35 30 30 25 20 15 10 Samples [n] 25 20 15 10 5 5 0 0 0,0-0,25 0,25-0,5 0,5-0,75 0,75-1,0 1,0-1,25 1,25-1,5 1,5-1,75 1,75-2,0 2,0-2,25 0,05-0,10 0,10-0,15 0,15-0,20 0,20-0,25 0,25-0,30 0,30-0,35 Theanine [%] Glutamic acid [%] Pu-Erh Tea (n = 3) Oolong Tea (n = 1) White Tea (n = 6) Green Tea (n = 27) Black Tea (n = 47) 24 Samples [n]

5 4 3 2 1 0 Contents of theanine and glutamic acid in first flush and second flush teas 5 4 3 2 1 0 25 0,3-0,6 0,6-0,9 0,9-1,2 1,2-1,5 1,5-1,8 0,08-0,13 0,13-0,18 0,18-0,23 0,23-0,28 0,28-0,33 Theanine [%] Glutamic acid [%] Samples [n] Samples [n] First Flush (n=7) Second Flush (n=4) First Flush (n=7) Second Flush (n=4)

6 5 4 3 2 1 0 Contents of theanine and glutamic acid in Japanese teas and Chinese teas 6 5 4 3 2 1 0,4-0,6 0,6-0,8 0,8-1,0 1,0-1,2 1,2-1,4 Theanine [%] 0 0,15-0,18 0,18-0,21 0,21-0,24 0,24-0,27 0,27-0,30 Glutamic acid [%] China (n = 11) Japan (n = 6) China (n = 11) Japan (n = 6) 26 Samples [n] Samples [n]

Behaviour of theanine and glutamic acid during the production of tea extract Raw material Extraction Filtration Concentration Pasteurisation 0 0,1 0,2 0,3 0,4 0,5 0,6 Contents [%] Theanine Glutamic acid based on the raw material 27

Summary Histamine in fish 12 samples can be analyzed within 6 hours Theanine in tea 24 samples can be analyzed within 8 hours 28

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Derivatisation 1 ml of the filtrate is adjusted to ph 8 Add 1 ml borate-puffer and 2 ml dansyl chloride solution Incubate for 1 h Adjust to 10 ml with water SO 2 CL O 2 S N H R + R-NH 2 - HCL N(CH 3 ) 2 N(CH 3 ) 2 Borate puffer Mix 67.3 ml of a 0.25 M borax solution with 32.7 ml of a 1 M boric acid, for preparing 100 ml you need 6.225 g boric acid and 9.525 g sodium borate 30

Asp+Gln Trennung der wichtigen AS Arg Glu The The, Arg, Asp, Glu und Gln weitere Aminosäuren: Elution in ausreichendem Abstand zu Theanine (außer Gly) Coelution von Glu und Thr Trennung der Aminosäuren Arg, Asp, Glu, Gln und The Asp Glu Arg Gln Thr Gly Met Cys Ser The Trp Ala Trennung eines Aminosäuregemisches 31

BAI = c Histamin + c Spermin Putrescin 1+ c + c + c Spermidin Cadaverin 32

Theanine biosynthesis NH 2 OH HOOC NH 2 O L-Glutamat- Ethylamin-Ligase NH 2 + + HOOC O N H H 2 O Glutaminsäure Ethylamin Theanin in den jungen Wurzeln der Teepflanze danach Transport in die jungen Blätter (Wickremasinghe et al. 1972) 33