Application Note

2006

Distillation Unit K-355

Determination of Total Volatile

Basic Nitrogen (TVB-N) in fish and
shrimps

Application No. K355-006, Version A Page 1

1 Introduction
This application note describes the determination of TVB-N in fish and shrimps.
Measurement of Total Volatile Bases (TVB) is one of the oldest chemical methods for
measuring freshness; it was first described in the early 1930's. In this application note a
fish sample or an extract of fish muscle is alkalined and the free bases are distillated.
The volatile bases are titrated with acid and the amount of acid is used to calculate the
total bases being distillated. The bases are a mixture of ammonia, trimethylamine and
dimethylamine, which each contain one basic nitrogen atom per molecule. TVB is usually
expressed as mg nitrogen/100 g of muscle tissue. The ammonia content of live fish is
negligible, (ammonia is quite toxic to fish), but is formed in the flesh soon after death due to
bacterial or enzymatic action on amino acids.

2 Compliance
The method fulfills the official regulation o 95/149/EC

3 Summary of method
A protein-free extract of fish muscle is prepared with 0.6 N perchloric acid. An aliquot of the
extract is taken, alkalinized with sodium hydroxide. The volatile nitrogenous bases are
distilled and trapped in boric acid. The concentration of the bases is determined by
titration with standard acid.

4 Preservation of samples
Investigate immediately, otherwise quick freeze the minced sample (e.g. in a closed
container/plastic bag) and store preferably at -25°C or lower.

5 Equipment
-Distillation Unit K-355 -Pipettes (10, 50, 100 mL)
-Mettler Toledo DL 22 F&B -Folded filter, diameter 150 mm,
-Mettler electrode DG 115-SC quick-filtering
-Mixer B-400, with PVDF Schleicher & Schuell, 595 1/2
as sealing membrane -Volumetric flasks (100 / 250 / 500 /
-Beaker 300 mL 1000 mL)
-Powder/Liquid funnels

Application No. K355-006, Version A Page 2

6 Chemicals

- Perchloric acid solution (6 g/100 mL), Fluka 08612

- Sodium hydroxide solution (20 g/100 mL), Fluka, 55973
- Boric acid solution (3 g/100 mL), Fluka, 55293
- Silicone anti-foaming agent, Fluka, 85390
- Phenolphthaleine solution, Fluka, 43607
- Hydrochloric acid 0.1 mol/L, Riedel-de-Haen, 35335
- Ammonium chloride, Fluka, 09700
- Tashiro Indicator, Fluka, 36083(for manual titration)

A kit for the determination of TVB in Fish, according EC guideline (95/149/EC), Fluka
66662 is commercial available.
The kit contains ready-to-use reagents for this method. The kit contains all reagents
needed for 10 determinations of TVB. The suitability of all 7 reagents was thoroughly
tested. The concentrations and specifications of perchloric acid solution, sodium hydroxide
solution and boric acid solution were especially tailor-made for this method which reduces
enormously the time required.

6.1 Solution preparation

Diluted hydrochloric acid solution 0.01 mol/L
Dilute hydrochloric acid 0.1 mol/L with water 10+90.

7 Procedure
Important safety aspect !

When working with perchloric acid, which is strongly corrosive, necessary caution and
preventive measures should be taken. Use PVDF as sealing material for the Mixer B-400.

7.1 Sample preparation

Take a fish sample of at least 100 g (preferably a total fillet) and homogenize throughly by
cutting. From frozen fish, e.g. fillet blocks, cut a 100-200 g sample of approx. 2 cm
thickness, place it in a water-tight plastic bag and thaw, e.g. by immersing the bag in a
gently stirred water bath at about 20°C but not more than 25°C; thaw time: approx. 15 min.
Bigger skin and bone pieces should be removed prior to homogenization.
Weigh 10.0 g from the well-homogenized fish flesh sample into the mixing vessel. Add
90.0 mL perchloric acid solution, blend for two minutes with a high-speed mixer and then
filter through a filter paper.
The extract thereby obtained can be kept for at least seven days at a temperature between
approximately 2°C and 6°C.

Application No. K355-006, Version A Page 3

7.2 Distillation settings

Instrument: K-355
Sample size 50 ml Receiver volume 100 mL
Steam power 90 % Distillate volume approx. 100 mL
Distillation time 4-5 min Condensate volume 130 – 150 mL
Receiver Boric acid
solution

7.3 Steam distillation

1. Prepare the K-355 by preheating the unit for 5 minutes.
2. Add 50 mL of the extract to the distillation tube. For a later check on sufficient
alkalinization of the extract, several drops of phenolphtalein are added. Add a few drops
silicone antifoaming agent.
3. Attach the distillation tube to the instrument.
4. 6.5 mL of sodium hydroxide solution are dosed to the distillation tube.
5. The distillate outlet tube is submerged in a receiver with 100 mL boric acid solution, to
which three to five drops of the indicator solution have been added.
6.Start the distillation.
A distillation volume of approx. 100 mL will be achieved with a steam setting of 90% and a
distillation time of 4-5 minutes. This will vary slightly from instrument to instrument and
depends on the sample type. The volatile bases contained in the receiver solution are
determined by titration with standard hydrochloric solution. The pH of the end point should
be 5.0 +/- 0.1.
Employ a minimum of one blank per sample batch to determine if contamination has
occurred.

8 Titration parameter
Titrator DL 22 F&B
Electrode Mettler Toledo DG 115-SC
Endpoint pH 5.0
Method Modified Mettler method 10:
Endpoint pH 4.6 changed to pH 5.0
Titrant 0.01 mol/l HCL
Calculation Consumption (mL)

Application No. K355-006, Version A Page 4

9 Calculation of TVB
TVB-N concentration expressed in mg/100 g sample is calculated with the following
equation:

(Vs − Vb) * 0.14 * 2 * 100

TVBN =
m

Legend
Symbol Text Unit
TVB-N Total Volatile Base mg/100
Vs Consumption 0.01 mol/lHCl sample mL
Vb Consumption 0.01 mol/lHCl blank mL
m Weight of sample g

10 Verification

10.1 Recovery rate measurements with standard solution

Recovery measurements were carried out with ammonium chloride as standard.
Three solutions at different concentrations were measured:
Solution I conc. 107.60 mg N/100 g
Solution II conc. 42.82 mg N/100 g
Solution III conc. 21.41 mg N/100 g

Results:

Sample Recovery solution I Recovery solution II Recovery solution III

(%) (%) (%)
1 99.26 100.37 98.50
2 100.36 99.95 100.56
3 99.73 99.58 98.80
4 100.08 99.63 100.75
5 100.25 99.49 99.44
Mean value 99.93 99.80 99.61
rsd (%) 0.44 0.36 1.01

Application No. K355-006, Version A Page 5

10.2 Recovery rate measurements in fish (Zander-Green-pike) after standard addition
The fish fillet was obtained from a local shop and had been stored in a refrigerator at 4-6°.
A fish extract solution was prepared (6.1) and aliquotes were spiked with standard solution
for analysis.

Fish extract conc. 13.11 mg N/100 g

Solution I conc. 107.60 mg N/100 g
Solution II conc. 42.82 mg N/100 g
Solution III conc. 21.41 mg N/100 g

Results:

Sample Recovery solution I Recovery solution II Recovery solution III

(%) (%) (%)
1 99.41 100.18 101.33
2 99.72 99.77 99.62
3 99.71 100.0 99.05
4 99.98 99.72 99.33
5 99.63 101.50 100.76
Mean value 99.69 100.23 100.02
Rsd (%) 0.20 0.73 0.98

10.3 Recovery rate measurements in shrimps after standard addition

The shrimps were purchased from a local shop and had been stored in a refrigerator at
4-6°. The shrimps were in a preservation liquid containing E 211(sodium benzoate), E 202
(potassium sorbate) and E 330 (citric acid). A shrimp extract solution was prepared (6.1)
and aliquotes were spiked with standard solution for analysis.

Shrimps extract conc. 5.73 mg N/100 g

Solution I conc. 107.60 mg N/100 g
Solution II conc. 42.82 mg N/100 g
Solution III conc. 21.41 mg N/100 g

Results:

Sample Recovery solution Recovery solution II Recovery solution III

I (%) (%)
(%)
1 99.30 99.17 99.05
2 99.84 99.35 99.65
3 99.25 99.39 100.94
4 99.61 99.03 99.16
5 99.30 99.30 99.70
Mean value 99.46 99.25 99.7
rsd (%) 0.26 0.15 0.75

Application No. K355-006, Version A Page 6

10.4 Typical TVB-N levels in stored fish

TVB-N quantity vs storage time

100
TVB-N (mg per 100 g sample)

90
80
70
60
50
40
30
20
10
0
0 5 10 15 20 25
Storage time (days)

Volatile base content of samples stored at refrigerated temperature ( 4-6°)

10.5 Remarks
During the first 8-10 days of storage, the concentration of TVB-N remains almost constant.
After this period of storage, TVB-N values begins to increase rapidly with time.
According to Connell,J.J, the fish is considered spoiled when TVB-N exceeds 30 mg
N/100 g fish.

EC guideline (95/149/EC):
Article 1
Unprocessed seafood products belonging to the species categories listed in Annex I shall
be regarded as unfit for human consumption where, organoleptic assessment having
raised doubts as to their freshness, chemical checks reveal that the following TVB-N limits
are exceeded:

1. 25 mg of nitrogen/100 grams of flesh for the species referred to in point A of Annex I;

2. 30 mg of nitrogen/100 grams of flesh for the species referred to in point B of Annex I;
3. 35 mg of nitrogen/100 grams of flesh for the species referred to in point C of Annex I.

ANNEX I
SPECIES CATEGORIES FOR WHICH A TVB-N LIMIT VALUE IS FIXED
A. Sebastes spp.
Helicolenus dactylopterus Sebastichthys capensis B. Species belonging to the
Pleuronectidae family (with the exception of halibut: Hippoglossus spp.) C. Salmo salar
Species belonging to the Merlucciidae family Species belonging to the Gadidae family
Note:
EU Commission Decision 95/149/EC defines maximum TVB contents only of few species
or families of fish above which the fish is considered unfit for consumption.

Application No. K355-006, Version A Page 7

11 References
1. EC guideline (95/149/EC)
2. Connell,J.J. Control and Fish Quality, Fishing News (Books) Ltd. Farnham, Survey,
UK,(1975)

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CH-9230 Flawil/Switzerland
T +41 71 394 63 63
F +41 71 394 65 65

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Application No. K355-006, Version A Page 8