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PH-Metric Determination of the Acid Value of Vegetable Oils without Titration

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 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999 915
FOOD COMPOSITION AND ADDITIVES
pH-Metric Determination of the Acid Value of Oilseeds
without Titration
PVM 1:1999
KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999
Abstract
A pH-metric method for determining the acid values (AVs) of
oilseeds in the range 0.6–10 mg KOH/g oil or more has been
developed. The method is based on rapid (1–2 min) and com-
plete extraction of free fatty acids from an oilseed test portion Peer-Verified Methods Program.
into a special reagent A, transfer of an aliquot of the reagent
into pH-metric cell with another reagent B, and then measure-
ment of the conditional pH in the mixture of reagents by a
glass electrode. Total time for the determination including cal-
culation is 9–10 min. The method is rapid, cheap, simple for
automation, and not labor intensive.
Method Authors
ILYA KUSELMAN, YAKOV I. TUR’YAN, TAT’YANA BOURENKO,
IRENE GOLDFELD, and AVINOAM SHENHAR
The National Physical Laboratory of Israel (INPL), Danciger
A Bldg., Givat Ram, Jerusalem 91904, Israel, Tel.:
+972-2-6536534, Fax: +972-2-6520797, E-mail:
kuselman@netvision.net.il
Submitting Laboratory
Materials Lab at The National Physical Laboratory of Israel,
Danciger A Bldg., Givat Ram, Jerusalem 91904, Israel
Peer-Verified Laboratory
Bio-Lab Ltd., Industrial Zone Atarot, PO Box 34038, Jerusa-
lem 91340, Israel
Acknowledgment of Reviewers
Technical Referee: David Firestone
Technical Reviewers: Charles Warner and Magdi Mossoba
Summary of Results of Verification Study
Submitting Laboratory
Validation parameters were evaluated for soybean, canola,
and sunflower oilseeds. In addition to 3 samples of initial pur-
chased oilseeds, 2 fortified samples of each oilseed were pre-
pared with additives of known amounts of oleic acid. A total
of 9 samples were studied. For each sample, the average of
10 AV determinations by the standard method (oil extraction
in soxhlet and titration) is taken as the “true” value. Four repli-
cates were analyzed in 1 day over a period of 5 days for all
samples by the verified pH-metric method. A total of 9 × 5 × 4
= 180 AV determinations were performed by the verified
method and 3 × 10 = 30 by the standard method. Evaluated ac-
curacy, recovery, repeatability, reproducibility, and limit of
quantitation satisfy the requirements of the AOAC
Peer Laboratory
The same 3 samples of purchased oilseeds were analyzed by
the verified method for evaluation of accuracy, repeatability,
and reproducibility. The results of AV determination by the
standard method obtained in the submitting laboratory were
used as “true” values. Samples were analyzed over a period of
5 days, 4 replicates daily; that is, 3 × 5 × 4 = 60 AV determina-
tions were performed by the verified method. All evaluated
parameters satisfy the requirements.
Safety Precaution
Solutions of a strong acid, H2SO4, are used: all operations should
be performed with protective safety glasses and gloves (1).
1. Scope
1.1. Introduction and Matrixes
International standards for determination of the acid value
(AV) of oilseeds are based on time-consuming (4 h and more)
extraction of the oil (2) and then acid-base titration of the free
fatty acids contained in the oil (3). The method described be-
low is based on rapid extraction (1–2 min) of the free fatty ac-
ids from the oilseeds with special reagents (4, 5) and
pH-metric AV determination of an aliquot of the extract (6–8).
Total time for the determination including calculation is
9–10 min. The method has the following advantages: rapidity,
simplicity, use of cheap instrumentation, and facility of auto-
mation.
This method is intended for determination of the AVs of such
oilseeds as sunflower, soybean, or canola during their cultiva-
tion and use as raw materials for oil production.
1.2. Applicable Range and Concentrations
The range of the AV determination is 0.6–10 mg KOH/g oil
(upper limit may be more).
1.3. Limitation
Limitations are unknown.
916 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999
2. References
(1) Garfield, F.M. (1992) Quality Assurance Principles for Ana-
lytical Laboratories, AOAC, Arlington, VA, pp 113–115
(2) ISO 659: 1988-02-15, Oilseeds—Determination of hexane
extract (or light petroleum extract), called “oil content”
(3) ISO 729: 1988-11-01, Oilseeds—Determination of acidity of
oils
(4) Tur’yan, Y.I., Berezin, O.Y., Kuselman, I., & Shenhar, A.
(1995) Israeli Patent Appl. 115677, 19.10.95
(5) Berezin, O.Y., Tur’yan, Y.I., Kuselman, I., & Shenhar, A.
(1996) J. Amer. Oil Chem. Soc. 73, 1707–1711
(6) Tur’yan, Y.I., Berezin, O.Y., Kuselman, I., & Shenhar, A.
(1994) Israeli Patent Appl. 110192, 01.07.94
(7) Tur’yan, Y.I., Berezin, O.Y., Kuselman, I., & Shenhar, A.
(1996) J. Amer. Oil Chem. Soc. 73, 295–301
(8) AOAC Peer-Verified Method 1:1997 “pH-Metric Acid Value
Determination in Vegetable Oils without Titration”
(9) Hui, Y.H. (Ed.) (1992) Encyclopedia of Food Science and
Technology, Vol. 3, p. 1901
(10) “Quantifying Uncertainty in Analytical Measurement,”
EURACHEM Guide, 1995
(11) Kuselman, I., & Shenhar, A. (1997) Accred. Qual. Assur. 2,
180–185
3. Definition
AV is the amount of potassium hydroxide, expressed in milli-
grams, required to neutralize the free fatty acids in 1 g of oil
extracted from oilseeds.
4. Principle
Two special reagents are used. Reagent A consists of a weak
base (triethanolamine; TEA) in a suitable solvent (a mixture of
heptane, isopropyl alcohol, and a little water). The TEA in the
volume for analysis should be in significant excess of the pos-
sible free fatty acid content of the oilseed test portion. The ex-
cess TEA allows solid–liquid extraction of the free fatty acids
from the oilseeds within 1–2 min:
(ΣHAni)seed + TEA → (ΣTEA × HAni)A (1)
where HAni is ith free fatty acid and (ΣTEA × HAni)A is the
corresponding TEA salt in reagent A, partly or completely dis-
sociated.
Reagent B includes TEA excess, as in reagent A; a solvent
(isopropyl alcohol and water, 1 + 1, v/v); and a salt to support
the ionic strength. The high water concentration in reagent B
allows liquid–liquid extraction of the TEA salts of the free
fatty acids from an aliquot of reagent A (i.e., from the
heptane–isopropyl alcohol phase) into the isopropyl alco-
hol–water phase within 1–2 min:
(ΣTEA × HAni)A → {(TEA)H+}B + {ΣAn–}B (2)
These salts change the pH of reagent B, because a buffer mix-
ture of TEA and {(TEA)H+}B is formed. The conditional pH1′
of the buffer mixture is measured with a commercial aqueous
reference Ag/AgCl electrode and a pH meter calibrated by
standard aqueous buffers. After a standard acid (H2SO4) is
added to the mixture, a second conditional pH2′ is measured.
The difference between the results of these 2 pH measure-
ments is used for calculation of the oil’s AV. The calculation
is based on the linear dependence of pH on the logarithm of
{(TEA)H+}B concentration with slope 1 at sufficient TEA ex-
cess (7).
The value of the oil content in oilseeds is essential for this cal-
culation and should be known from previous information (5).
If such information is absent, data in the Encyclopedia of Food
Science and Technology (9) can be used for a first approxima-
tion: for example, oil contents of soybean, canola, and sun-
flower oilseeds are 20, 40, and 47%, respectively.
5. Standard
5.1. Purity and Dilution
A 0.1N solution of sulfuric acid is used as a standard acid (1)
for suitability test of the pH-metric system and (2) for addition
to the reaction mixture. This solution is prepared by diluting
H2SO4 with deionized water in a 1 L volumetric flask (7.16).
5.2. Stability
The standard (0.1N H2SO4) is stable at room temperature for
3 months.
6. Reagents and Supplies
6.1. Reagent A (4).—Prepared by mixing 500 mL TEA so-
lution (6.7) with 375 mL n-heptane (6.6).
6.2. Buffers.—With pH of 7.00 ± 0.02 and 9.22 ± 0.02 at
20°C.
6.3. Sulfuric acid.—CAS 7664-93-9, 0.1N.
6.4. TEA.—CAS 102-71-6.
6.5. Isopropyl alcohol.—CAS 67-63-0.
6.6. n-Heptane.—CAS 142-82-5.
6.7. TEA solution.—Prepared by dissolving 7.46 g TEA in
500 mL isopropyl alcohol–water, 97.5 + 2.5, v/v.
6.8. Reagent B (6).—Prepared by dissolving 1.0 g KNO3
and 29.8 g TEA in 1 L water–isopropyl alcohol, 50 +
50, v/v. To determine initial pHN value of the reagent
(pHN0), add 2.0–2.5 mL 0.1N H2SO4 to the 40–50 mL
reagent and titrate against a solution of 0.1N KOH in
the reagent (6.9) by potentiometry. The electrodes for
the titration (a glass electrode and a reference elec-
trode) should be the same as the ones used for AV de-
termination. The equivalence point of the titration cor-
responds to pHN0 of the reagent. As a rule, the initial
reagent is acidic, that is, the pH′ of the reagent is
lower than pHN0 because of contact with atmospheric
CO2. Thus, it is necessary to add to the reagent a small
 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999 917
amount of 0.1M KOH until pHN = pHN0. The prepared
reagent should be maintained in a bottle protected
from CO2.
6.9. Potassium hydroxide (0.1N) in reagent B (6.8).—Pre-
pared by dissolving KOH (6.10) in the reagent.
6.10. Potassium hydroxide pellets.—CAS 1310-58-3.
6.11. Oleic acid.—CAS 112-80-1.
6.12. Oleic acid (0.1M) in heptane.—Prepared by dissolv-
ing 28.247 g oleic acid (6.11) in 1 L n-heptane (6.6).
6.13. Deionized water.—With specific conductivity not to
exceed 1 × 10–4 S/M (simens/metre) at 20°C.
7. Apparatus
7.1. pH meter.—With uncertainty of ±0.01 pH unit.
7.2. Glass working electrode and reference Ag/AgCl elec-
trode.
7.3. pH-metric cell.
7.4. Magnetic stirrer for pH-metric cell.
7.5. Calibrate pH meter once a day at the beginning of the
work.—The calibration is carried out in the pH-metric
cell with 2 buffer solutions (6.2).
If the pH-metric system is unstable (drift of the results
of the pH measurement or their nonsystematic
changes), the glass electrode (7.2) should be cleaned.
For this purpose, the electrode is introduced and held in
the pH-metric cell with isopropyl alcohol and magnetic
stirrer for 3–5 min. Then the electrode is cleaned
2–3 times in the same cell with deionized water, each
time for 2–3 min. The clean electrode should be kept in
0.05N HCl for 10–15 h before use.
System suitability is tested by the dependence of condi-
tional pH on the logarithm of the concentrations of the
standard (sulfuric) acid added in reagent B. With this
aim, 100, 100, 200, 400, and 800 µL of 0.1N H2SO4
(6.3) are added to 50 mL reagent B in the pH-metric cell
and corresponding pH1N, pH2N, pH3N, pH4N, and pH5N are
measured. The test is successful if the differences
pH1N–pH2N, pH2N–pH3N, pH3N–pH4N, and pH4N–pH5N are
0.30 ± 0.02.
The test should be done at least once a week, even when
a drift or nonsystematic changes in the work of the
pH-metric system are imperceptible.
7.6. Beaker.—50 mL.
7.7. Petri dish.—90 mm diameter.
7.8. Disposable test tube.—For centrifugation, graduated,
50 mL capacity.
7.9. Mechanical hand pipetters.—Capacities up to 5 mL.
7.10. Analytical balance.—With uncertainty of ±0.0002 g.
7.11. Centrifuge.
7.12. Mixer.
7.13. Cofee grinder.
7.14. Drying oven.—±2°C.
7.15. Cylinder.—Graduated, 50 mL capacity.
7.16. Volumetric flask.—1 L, class A according to Deuthche
Industrie Norm (DIN) 12664.
8. Sampling
8.1. Procedure
The sampling procedure is standard (2). The oilseed sample to
be analyzed should not have less than 4–5 test portions (11.1)
and should be well mixed.
8.2. Storage
Oilseed samples in sealed bottles are stored in a refrigerator,
where AV changes are negligible for a period of 6 months.
9. Sample Preparation
Moist oilseeds (when moisture is >7.5–8.5%) should be dried
in a drying oven (7.14) at 70°–80°C for 1–2 h or, preferably, at
50°–60°C overnight. Dried oilseeds are ground in the coffee
grinder (7.13) until they look like a homogeneous meal.
10. Preparation of Controls
10.1. Blanks
Determination of AV is carried out without blanks.
10.2. Control Samples
A fortified sample of the oilseed or a sample previously ana-
lyzed by the standard method (3) is used as a control sample.
10.3. Fortification
A fortified sample is prepared from an ith test portion (Gi, g)
of the sample analyzed by the present method (initial sample).
Oleic acid solution (6.12) is added to this test portion in the
test tube before introduction of reagent A (11.2). The volume
of oleic acid solution (Voa, mL) should be calculated with the
assumption that the AV of the fortified sample (AVf) is
3 times greater than AV of the initial sample (AVi; AVf =
3AVi):
Voa = 2 × 1000 × Gi × P × AVi /
[Coa × Moa × (199 – 3 × AVi)]
= 70.8 × Gi × P × AVi/(199 – 3 × AVi) (3)
where P is oil content in the oilseed (parts of 1), Moa and Coa
are the molecular weight (g) and concentration (0.1M) of oleic
acid, and 199 is the ratio between molecular weights of potas-
sium hydroxide multiplied by 1000 and oleic acid.
918 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999
For example, if P = 0.2, AVi = 1 mg KOH/g oil and Gi = 7 g,
the recommended Voa is 0.5 mL according to equation (3).
Although fortification is rapid, use of a fortified sample can-
not ensure control of solid–liquid extraction. For this purpose,
a sample analyzed by the standard method (3) is the suitable
control.
11. Procedure
11.1. Test Portions
The test portion is put into a previously weighed disposable
test tube for centrifugation (7.8). The test tube with the oilseed
is weighed again. The mass of the test portion is the difference
between the 2 test tube weighings. Recommended masses of a
test portion for oilseeds with minimal oil content P = 0.2 are
shown in Table 1 (a greater mass corresponds to a lower ex-
pected AV). If P > 0.2, the recommended mass in Table 1 is
decreased in P/0.2 times.
11.2. Extractions
Reagent A (30 mL) is added to the disposable test tube with an
oilseed test portion. The test tube is closed by own plug and
shaken vigorously with a mixer (7.12) or by hand for 1–2 min.
The test tube is centrifuged for 1–2 min for phase separation
(filtration can be used for this purpose too). An aliquot of the
upper liquid phase (5–20 mL) is removed with a mechanical
pipetter and transferred to the pH-metric cell. Reagent B
(50 mL; 6.8) is added to the cell, and the stirrer is turned on to
provide good mixing of the components and to ensure extrac-
tion of the TEA salts of the free fatty acids from the aliquot
into reagent B.
11.3. Measurements
The electrodes should be introduced into the cell with re-
agent B and the aliquot, and after 1–2 min, the stable pH1N is
read. Then a certain volume (e.g., 0.2 mL) of standard 0.1N
H2SO4 solution is added with stirring for 1 min, and the stable
pH2N is read. The optimal standard acid addition should ensure
that )pH = pH1N – pH2N . 0.25–0.35.
12. Calculations
12.1. AV
The AV of the oilseed sample is calculated according to the
following formulas:
Table 1. Mass of a test portion
Expected AV, mg KOH/ g oil Mass, g
0.6–2 5–10
2–5 2–5
5–10 1–2
>10 <1
AV = AVal × V/Val
and
AVal = (56.11 × Nst × Vst)/[G × P × (10)pH – 1)] (4)
where AVal is the acid value corresponding to the aliquot of
reagent A used for pH measurements (mg KOH/g oil), V is the
volume of reagent A introduced into the test tube (30 mL), Val
is the volume of the aliquot used for pH measurements (mL),
56.11 is the molecular weight of KOH, Nst is the concentration
of the standard H2SO4 solution (0.1N), Vst is the volume of the
standard acid added (mL), G is the mass of the oilseed test por-
tion (g), and G × P is the analyzed oil mass (g).
12.2. Uncertainty
The AV confidence interval at the 95% level of confidence is
AV ± U, where U is the expanded uncertainty (10):
U = 2 × AV × {[u(AVal)/AVal]2 + [u(V)/V]2
+ [u(Val)/Val]2}1/2 (5)
In equation 5, the parameter 2 is the coverage factor; the stan-
dard uncertainty u(AVal)/AVal = 0.07 (11), and u(V)/V and
u(Val)/Val are negligible in comparison with U(AVal)/AVal.
So, U = 0.14 AV.
13. Test Results Report
Results of AV determination and its confidence interval AV ±
U(AV) should be rounded and reported with only one signifi-
cant digit after the decimal point; for example, 1.1 ±.0.2, 4.5 ±
0.6, 10 ± 1.
14. Quality Assurance
14.1. Critical Control Points
The following critical control points should be taken into ac-
count for successful determination of AV by the pH-metric
method:
(1) An oilseed test portion and reagent A should be well mixed
for complete solid–liquid extraction. (2) The solid phase
should be separated completely from the liquid phase. Pres-
ence of oilseed particles in reagent B dramatically increases
the uncertainty of the result. (3) The aliquot of reagent A
should be large enough to decrease the pH of reagent B in the
pH-metric cell by 0.5–0.6 pH unit (pH0N – pH1N > 0.5) to pro-
duce a readable analytical signal. (4) pH values during the
reading should be stable. (5) )pH should be in the range
0.25–0.35 for minimum uncertainty.
14.2. Analysis of Control Samples
AV determination should be performed once a day for the con-
trol (fortified) sample prepared according to 10.3. The determi-
nation will be under control if the AV obtained for the control
sample (AVc) corresponds to the following requirement:
|AVc – AVc c|/AVc ≤ 21/2 U(AVc )/AVc = 0.20 (6)
 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999 919

where AVcc is the calculated value of AVc (equation 3):
AVcc = (AVi × Gi × P + 56.11 × Voa × Coa)/(Gi × P) (7)
When the sample analyzed by the standard method is used as a
control, the procedure is under control if the difference be-
tween the results of analyses by the 2 methods is less than
U(AVc) = 0.14 (i.e., the uncertainty of the standard method is
negligible).
Report of INPL (The Submitting Laboratory):
Within-Laboratory Validation of the New Method
for pH-Metric Acid Value Determination
in Oilseeds without Titration
15. The Program of the Experiment
15.1. Determination of AV by the standard method in the
purchased canola, soybean, and sunflower oilseeds,
10 replicates for each oilseed.
15.2. Fortification of oilseed test portions by addition of cal-
culated volumes of 0.1M oleic acid into test tube. The
calculation is fulfilled by equation 3 in the text of the
validated method prepared in ISO format, where in-
stead of the coefficient of fortification 3, another one
can be used: 1.5, 2, or 6. The first sample of canola
oilseed fortified in this way is intended to have an AV
that is 1.5 times higher than the AV of the initial (pur-
chased) oilseed. The second fortified sample is in-
tended to have an AV that is 2 times higher than the
AV of the initial oilseed. For soybean and sunflower
oilseeds, fortification should ensure 3 and 6 times
higher values of AV compared with AV of the initial
oilseeds for the first and second fortified samples, re-
spectively.
15.3. Determination of AVs of purchased oilseeds and for-
tified samples by the pH-metric technique, 4 replicates
for each sample daily over a period of 5 days.
16. Results and Discussion
Average results from 10 replicate determinations by the stan-
dard titration method, used as “true” AV values, are given in
Tables 2–4. For fortified samples, “true” values were calcu-
lated according to equation 3. Results of pH-metric determi-
nations and corresponding statistical data are also given in Ta-
bles 2–4.
16.1. Precision: Repeatability, Intermediate
Precision, and Reproducibility
For all samples, values of the replicate relative standard devia-
tion RSD1 (during a day) characterizing repeatability and val-
ues of the daily relative standard deviation RSD2 (between
days) characterizing intermediate precision satisfy Horwitz’s
criterion. The same is true for the data of the independent labo-
ratory. Between-laboratory deviation (INPL–Bio-Lab Ltd.),
characterizing reproducibility, is also acceptable because the
biases of the laboratory average results from the correspond-
ing “true” values are negligible (16.2). Therefore, repeatabil-
ity and reproducibility are satisfactory. Moreover, variations
of reagents, equipment, analysts, standards, time, and other
conditions were inevitable during this method trial. Therefore,
all the experiments also can be interpreted as the method rug-
gedness testing. The result of this testing is satisfactory be-
cause the precision criteria discussed above are satisfied.

Table 2. Determination of canola oilseed AVs by the method to be validated

AV, mg KOH/g oil, determined on indicated day
“True” Replicate t(0.95) ×
Sample value No. 1 2 3 4 5 RSD1, % RSD2, % Bias, % RSD2, %

Purchased 4.68 1 4.89 4.66 4.92 4.74 4.82 2.05 0.95 1.47 2.63
2 4.84 4.89 4.84 4.70 4.73
3 4.59 4.80 4.97 4.69 4.55
4 4.65 4.74 4.56 4.68 4.72
Fortified 1 7.02 1 7.19 7.29 7.26 7.39 6.76 2.56 1.83 0.74 5.09
2 6.85 7.04 7.20 7.32 6.82
3 6.78 7.18 6.75 7.08 6.79
4 6.93 7.28 7.47 6.86 7.20
Fortified 2 9.36 1 9.69 8.99 9.00 9.04 8.97 2.57 1.57 –1.47 4.36
2 9.45 9.23 9.00 8.79 8.94
3 9.08 9.47 9.08 9.44 9.28
4 9.76 9.08 9.78 9.04 9.33
920 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999

Table 3. Determination of soybean oilseed AVs by the method to be validated

AV, mg KOH/g oil, determined on indicated day
“True” Replicate t(0.95) ×
Sample value No. 1 2 3 4 5 RSD1, % RSD2, % Bias, % RSD2, %

Purchased 1.01 1 0.95 1.00 0.99 0.99 1.02 3.61 1.53 –2.67 4.25
2 0.96 1.02 0.98 0.99 1.04
3 1.03 1.01 0.99 1.00 0.92
4 0.90 0.90 0.97 1.05 0.95
Fortified 1 3.03 1 3.19 3.33 3.01 3.07 3.16 3.18 3.38 7.10 9.39
2 3.31 3.26 3.29 3.19 3.39
3 3.35 3.37 3.28 3.03 3.22
4 3.51 3.42 2.96 3.07 3.49
Fortified 2 9.09 1 9.55 9.70 9.43 9.03 8.88 2.97 2.95 3.10 8.20
2 9.92 10.15 9.44 8.82 9.29
3 9.56 9.97 8.84 9.52 9.35
4 8.99 9.58 8.65 9.35 9.41

16.2. Accuracy 16.3. Recovery

Recovery was determined for all fortified samples as follows:

Accuracy was evaluated as the bias of some average
pH-metric results from corresponding “true” values calcu-
lated as a relative percentage. Bias values, having positive as
well as negative signs, are less than the critical ones: t(0.95) ×
RSD2 at the 95% level of confidence and 4 degrees of free-
dom. Thus, the differences of results obtained by the standard
titration method and those by the pH-metric method are insig-
nificant, and accuracy is satisfactory. The same is true for the
data of the independent laboratory (Table 5).
R, % = (AVf – AVi) × Gi × P × 100/
(Voa × Coa × 56.11) (8)
where AVf and AVi are the average AVs for fortified and ini-
tial oilseed samples, respectively, determined by the
pH-metric method (see Tables 2–4; mg KOH/g oil); Gi and P
are the mass (g) of the oilseed test portion and its oil content
(parts of 1), respectively; and Voa and Coa are the volume (mL)
and concentration (M), respectively, of oleic acid added.

Table 4. Determination of sunflower oilseed AVs by the method to be validated

AV, mg KOH/g oil, determined on indicated day
“True” Replicate t(0.95) ×
Sample value No. 1 2 3 4 5 RSD1, % RSD2, % Bias, % RSD2, %

Purchased 1.01 1 1.06 1.11 1.04 0.98 1.04 4.39 2.42 3.12 6.72
2 0.90 1.04 1.09 1.07 0.98
3 1.02 1.06 1.04 1.10 1.09
4 1.02 1.09 0.95 1.06 1.09
Fortified 1 3.03 1 3.15 3.24 3.36 3.00 3.19 3.79 2.09 2.87 5.82
2 2.98 3.07 3.04 2.97 3.17
3 3.08 3.40 3.28 3.10 3.04
4 3.35 3.06 2.94 2.94 2.98
Fortified 2 9.09 1 9.98 9.23 9.98 8.72 9.82 2.61 3.24 5.47 9.02
2 9.65 9.82 9.65 9.35 9.63
3 9.86 9.49 9.86 8.60 10.11
4 9.98 9.10 9.98 9.58 9.35
 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999 921

Table 5. Statistical analysis of the results of AV determination in purchased oilseeds in the independent laboratory
by the method to be validated
Day/result of determination, mg KOH/g oil
“True” Replicate t(0.95) ×
Sample value No. 1 2 3 4 5 RSD1, % RSD2, % Bias, % RSD2, %

Canola 4.68 1 4.71 4.58 4.67 4.63 4.63 1.51 0.62 –0.68 1.71
2 4.51 4.72 4.53 4.69 4.65
3 4.73 4.49 4.59 4.64 4.70
4 4.64 4.59 4.83 4.69 4.74
Sunflower 1.01 1 1.01 0.96 0.95 0.94 0.98 3.13 2.76 –1.04 7.67
2 1.04 1.06 0.98 0.97 0.98
3 1.05 1.03 0.98 1.01 1.03
4 1.05 0.95 0.93 1.00 1.09
Soybean 1.01 1 1.03 1.00 1.03 1.03 0.99 1.19 0.82 –0.25 2.27
2 1.02 1.03 1.00 1.02 0.99
3 1.00 1.03 1.00 1.00 1.00
4 1.00 1.00 0.99 1.00 0.99

Average recoveries values (Table 6) were calculated from the
data in Tables 2–4, for 4 × 5 = 20 results of analysis. Oil con-
tents (P) for canola, soybean, and sunflower oilseeds are 0.40,
0.16, and 0.58, respectively; Coa = 0.1131M. Because recov-
ery is an additional characteristic of accuracy, which in our
case is satisfactory (see 16.2), the obtained recovery values
should be accepted as satisfactory also.
16.4. Limit of Quantitation
The limit of quantitation was determined from the dependence
of pHN on lg Na in reagent B (Na is the concentration of the
sum of acids in the reactive mixture, equivalents/L). The lin-
ear range of this dependence that we obtained (Figure 1) was
Na = 2.0 × 10–4M to 3.1 × 10–3M. Practically the same depend-
ence was found in the mixture of reagent B (50 mL) with an
aliquot of reagent A (up to 20 mL). The dependence corre-
sponding to the 20 mL aliquot is shown in red in Figure 1. At
Na < 2.0 × 10–4M, deviation from the linear dependence oc-
curs. This deviation can be explained by the increasing hydro-
lysis (solvolysis) influence of TEA (B) according to
equation 9:
B + H2O ø BH+ + OH– (9)
From the lower limit of Na in the linear range of the depend-
ence (2.0 × 10–4M), it follows that the limit of AV quantitation
is AVLOQ = 0.6 mg KOH/g oil for a 10 g maximum mass of an
oilseed test portion with an oil content of 16% and a maximum
aliquot of 20 mL for reagent A. This limit of quantitation is suf-
ficient to characterize oilseeds (usually AV $ 1 mg KOH/g oil).
16.5. Other Validation Parameters
Validation parameters such as calibration curve, sensitivity,
and specificity are not relevant for the validated method. The
study of the limits of detection and determination/decision
was not worthwhile because ready-to-use oilseeds do not have
AV < 0.6 mg KOH/g oil which was stated above as the limit of
quantitation.

Table 6. Determination of recovery from fortified samples (average for 20 oilseed test portions)
Oilseed AVi, mg KOH/g oil Gi, g Voa, g AVf, mg KOH/g oil Recovery, %

Canola fortified 1 4.75 2.1998 0.333 7.07 101

Canola fortified 2 4.75 2.0826 0.641 9.22 94
Soybean fortified 1 1.01 3.4655 0.179 3.25 108
Soybean fortified 2 1.01 1.5294 0.326 9.37 99
Sunflower fortified 1 1.04 4.4526 0.822 3.12 103
Sunflower fortified 2 1.04 4.5208 3.548 9.38 96
922 KUSELMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 4, 1999

Figure 1. Dependence pH¢ on lg Na for reagent B (line 1) and for the mixture of reagents B and A (line 2).

16.6. Stability of Novel Reagents
Reagent A used in the validation process (during 1 month) did
not have any influence on the validation data because it con-
tains TEA in large excess to free fatty acids in an oilseed test
portion, and a stable solvent (heptane + isopropyl alcohol +
water). Partial decreasing of the free TEA concentration by
contact with atmospheric CO2 is not important in this case. So,
reagent A can be stored in sealed vessels at room temperature
at least 1 month.
We assessed the stability of reagent B during validation of
PVM 1:1997 “pH-Metric Acid Value Determination in Vege-
table Oils without Titration.” The reagent can be used for
1 year if stored in a sealed container in an inert atmosphere.

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