1+ ENVIRONMENT CANADA

FISHERIES AND MARINE SERVICE

TECHNOLOGY BRANCH HALIFAX~ NOVA SCOTIA
.. E.G. BLIGH~ DIRECTOR

NEW SERIES CIRCULAR ~m. 61 NOVEMBER~ 1976

AN IMPROVED TITRIMETRIC METHOD FOR DETERMINATION
OF FREE FATTY ACID IN FISH OILS
- P.J. Ke, A.D. Woyewoda, L.W. Regier, and R.G. Ackman -

The free fatty acid (FFA) content, a continuing concern of workers

assessing the quality of oils and fats, has been widely used as a routine

quality index for fresh fish and related frozen and processed products.

The American Oil Chemists' Society (AOCS) method (1) for determination

o of FFA has often proven unsatisfactory for fish oils which are yellow or

red due to carotenoids, or brownish due to poor quality. Furthermore,

the slight turbidity in the marine oils and the ethanol-aqueous NaOH

titration system combined with endpoint fading, often masks the endpoint

color of the phenolphthalein indicator. C~spite special precautions,

such as multiple determinations, great difficulty may still be experienced

in obtaining consistent and accurate values for the FFA content of some

fish oil samples when using the AOCS or similar methods (2,3). A revised

titrimetric method has been developed for the determination of FFA content
~ ,

of oils which gives precise and accurate resul.ts ~ven when oils are highly

colored or rancid. The method is based upon the use of a mixed solvent

and meta cresole purple (MCP) as the indicator.

o
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( EXP ERIMEN'l'AL

A. Reagents:

Solvent Mixture: Chloroform, methanol and isopropanol (ACS grade)

mixed in a volume ratio of 2:1:2, is used for

dissolving the oil sample in. the titration.

Indicator: Meta Cresole Purple (MCP), obtained from Fisher

Sci. Co. and dissolved in distilled water at 0.5%

concentration, is used as the indicator.

Titrant: 0.05N NaOH in aqueous solution is used.

B. Procedure:

About 1 g of fish oil is accurately weighed < 0.005 g) into a 125

ml Erlenmeyer flask. To this is added solvent mixture (75 ml) and 4

drops of MCP indicator solution. While the dissolved sample is being

( mixed on a magnetic stirrer, the free fatty acids are titrated to the

purple end point with 0.05N NaOH using a 10 ml burette. A blank titra-
c .(.1sJl/
tion should be about 0.1 ml of ~ NaOH and should run daily.

C. Calculation:

The percentage of free fatty acids in most fish oils is calculated

as oleic acid from the following formula:

(T-B) x 1. 41
FFA, % in oleic
S
{l.d.N
where T and B denote the volume (ml) of ~ NaOH used to titrate the

sample and the blank, respectively, and S the weight (g) of sample.

RESULTS AND DISCUSSION

A comparison of the determination of FFA in three kinds of fish oils

by the AOCS and the MCP method described above is given in Table I. The

recommended multiple solvent system and MCP as indicator is clearly more

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( specific and reproducible than the AOCS titrimetric method procedure for

determining the content of FFA in fish oils. At concentrations of FFA

up to 13%, the results (Table I) obtained by the recommended MCP method

are slightly higher than the data from AOCS, but with a much lower standard

deviation of 1.5% or less. Inexperienced operators had no difficulty in

using the MCP method where in with the AOCS method there was inevitably

extra time spent on titrations due to the indecipherable color change of

phenolphthalein. The method described has been applied rapidly and

successfully to estimate the FFA contents of the various types of fish

oils shown in Table II. These samples included highly pigmented salmon

and redfish oils.

The multiple solvent system chosen for the recommended titrimetric

method for the FFA determination was a mixture of chloroform/methanol/

o isopropanolol ratio of 2:1:2 by volume. This solvent mixture will

completely mix with more than 12 ml of aqueous NaOH titrant before

becoming turbid. Furthermore, this solvent mixture can be simply prepared

by adding isopropanol and methanol to the chloroform extractant from

Bligh and Dyer's method of lipid recovery (4). A direct method for

estimating the content of FFA in a fish meat sample without removing or

changing the solvent from the extractant is being developed from the

new system of solvents and indicator.

The MCP endpoint, from yellow to purple at pH 12, was found to be

the most suitable in the newly developed titrimetric method. Other

indicators such as phenolphthalein (colorless to pink) and alkaline blue

(blue to orange-red), are subject to various degrees of undesirable

,

o color interference from the fish oil color interference at their endpoints.

Since the titration problems caused by color interference and turbidity

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( were eliminated, as little as one gram of oil (about 1/10 that used in

AOCS procedure) gave more reliable FFA results in various fish oils

(Table II).

Since the MCP titrimetric method for FFA analysis has advantages

over the AOCS procedure such as a greater reproducibility, a sharper

endpoint, a smaller sample size and superior miscibility of sol~ents,

it can be employed by various fish processing industries for the

determination of the FFA content in fish oils, particularly in highly

colored and oxidized samples.

REFERENCES

1. AMERICAN OIL CHEMISTS' SOCIETY. 1974. Official and Tentative

Methods of AOCS, 3rd edition, ca. 5a-40.

2. NERING, B. and MODZELEWSKA, K. 1975. Factor affecting the results

of fatty acid determination in various oils. Tluszcze Jadalne

19 : 129-136.

3. HORWITZ, W. 1976. Methods of analysis approved by the Codex

Alimentarius Commission. I. Acid Value. J. Assoc. Off.

Anal. Chern. 59 : 658-661.

4. BLIGH, E.G. and DYER, W.J. 1959. A rapid method of total lipid

extraction and purification. Can. J. Biochem. Physiol. 37 : 911.

Table I. Comparison of the determination of FFA in fish oils by the AOCS

and the recommended MCP method.

\ FFA AOCS Method * MCP Method *

Sample added \ FFASD \ RSD \ FFASD \ RSD

Herring oil 1.58 .06 3.8 1.60 .02 1.2

0.59 2.05 .09 4.4 2.13 .02 0.9

10.10 11. 67 .25 2.2 11.64 .06 0.5

Oxidized herring oil 2.58 .14 5.4 2.63 .04 1. 5

5.10 7 . 30 .27 3.7 7.60 .08 1.1

10.22 12.38 .25 2.0 12.85 .10 0.8

Redfish oil 2.90 2.87 .11 3.8 2.92 .02 0.7

12.49 12.33 .36 2.9 12.52 .04 0.4

* All data were calculated from 7 determinations.

S.D. and \ RSD are the standard deviation and percentage of relative

standard deviation, respectively.

(
Table II. The content of" FFA in several fish oils determined by the

recommended MCP method.

Fish Oils % FFA % RSD

Cod liver 3.34 .01 0.3

6.40 .02 0.3

Seal 2.35 .02 0.8

5.26 .03 0.6

Cod frame 1.98 .01 1.0

Salmon 2.56 .03 1.2

5.54 .04 , 0.7

Herring 4.61 0.1 0.2

Redfish 5.41 .03 0.7

0 Mackerel Frame 3.20 .05 1.5
8.42 .11 1.3

Mackerel skin 3.57 .01 0.3

Mackerel meats 2.70 .03 1.1

Oxidized herring 5.67 .06 1.1

Oxidized redfish 3.92 .06 1.5

Oxidized mackerel frame 4.81 .07 1.4

All data were calculated from 7 determinations.