Food Chemistry 81 (2003) 613–619

www.elsevier.com/locate/foodchem

Analytical, Nutritional and Clinical Methods Section

Spectrophotometric determination of total phosphorus in rape seeds

and oils at various stages of technological process: calculation of
phospholipids and non-hydratable phospholipids contents in
rapeseed oil
Aleksandra Szyd•owska-Czerniak, Edward Sz•yk*
Faculty of Chemistry, Nicholas Copernicus University, 87-100 Toruñ, Poland

Received 3 September 2002; received in revised form 30 November 2002; accepted 30 November 2002

Abstract
A new spectrophotometric method for determination of total phosphorus in rape seeds and rapeseed oils is described. The pro-
cedure is based on formation of the ionic—associate of molybdophosphate with Malachite Green (MG) in an acidic medium and
compared with the standard vanadomolybdate and molybdenum blue methods. Satisfactory values of recovery ranged between
99.1–100.3% and RSD < 1%, demonstrate the benefit of using MG method for the routine analysis of total phosphorus in oilseeds,
crude and refined oils. The equivalent of phospholipids contents in the crude and hydrated oils were calculated from the phospho-
rus determinations. Obtained results are discussed in respect of accuracy and precision.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: UV-vis spectrophotometry; Phosphorus; Phospholipids; Rape seeds; Rapeseed oil

1. Introduction Pérez-Camino, 2000; Dobson & Deighton, 2001;

Phospholipids (PL) in rapeseed oil are considered as
undesirable impurities causing refining problems and oil
losses. There are two chemically different sources of
phosphorus: inorganic phosphates and PL in edible oils.
Generally the total phosphorus content in oils is deter-
mined spectrophotometrically and then converted to
equivalent PL value.
Crude oil from rapeseed, which has been damaged in
the field, during storage or handling and transportation,
contains significant amounts of nonhydratable phos-
pholipids (NHP). These are PL which during the
degumming of crude rapeseed oil do not hydrate with
water, swell, form gels or precipitate from oil and are
not separated by centrifugation.
Among many techniques used in PL determination
the chromatographic methods are widely used for PL
analysis in different matrices, ranging from animal and
plant tissues, to seed oil extracts (Cert, Moreda, &
* Corresponding author.
E-mail address: eszlyk@chem.uni.torun.pl (E. Sz•yk).
0308-8146/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0308-8146(02)00562-9
Ostrowska, Skrzydlewska, & Figaszewski, 2000;
Rozentsvet, Dembitsky, & Saksonov, 2000; Singleton,
Ruan, Sanford, Haney, & Stikeleather, 1999; Touch-
stone, 1995). Among them HPLC and TLC are the
leading techniques for separation and quantitative ana-
lysis of PL in vegetable oils and lecithins, but quantita-
tive determinations can be affected by errors (Carelli,
Brevedan, & Crapiste, 1997; Chapman, 1980; El-Sebaiy,
El-Mahdy, Moustafa, & Mohamed, 1980; Kang & Row,
2001; Melton, 1992; Mounts, Abidi, & Rennick, 1992;
Nomikos, Karantonis, Fragopoulou, & Demopoulos,
2002; Nzai & Proctor, 1998; Racicot & Handel, 1983).
Fast, nondegradative and accurate analysis of PL in
edible oils and lecithins without prior separation is pos-
sible with 31P NMR (Bosco, Culeddu, Toffanin, & Pol-
lesello, 1997; Diehl, 2001; Kanamoto, Wada, Miyajima,
& Kito, 1981; Sotrihos, Herslöf, & Kenne, 1986).
Chromatographic techniques appear to be useful for
isolation and identification of PL mixtures. However
NMR spectroscopy is an accurate and precise method
for qualitative and quantitative analysis of the trace
amounts of PL in seeds, oil and lecithin samples.
614 A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619
Application of these techniques is not necessary, when
the total phosphorus content in studied material is in
the order of few hundred mg/kg. Therefore spectro-
photometric technique can be used as a method of
choice to the determination of PL in products of the
vegetable oil industry. Colorimetric methods were pre-
viously used for determination of total content of PL in
edible oils. Totani, Pretorius, and du Plessis (1982) and
Goh, Tong, and Gee (1984) reported that PL are
extractable from the vegetable oils and can be deter-
mined by the phospholipid–prussian blue complex in
glacial acetic acid and phospholipid–molybdenum blue
complex in hexane, respectively. Most of these proce-
dures are troublesome because pre-extractions of
molybdophosphate are needed to avoid large reagent
blanks. Moreover, these two methods are not directly
applicable, mainly due to low levels of PL in the refined
oil and the interference caused by colouring pigments
present in vegetable oils.
Therefore in the presented paper spectrophotometric
method of total phosphorus determination, based on
formation of the ion-associate of molybdophosphate
with Malachite Green (MG) was described. Altmann,
Fürstenau, Gielewski, and Scholz (1971) reported the
mechanism of the colorimetric reaction between molyb-
dophosphate and MG. Proposed method and standard
methods (PN-88/A-86930 and IUPAC 2.421) were
applied for quantitative determination of the total
phosphorus in seeds and rapeseed oil at different stages
of conventional technological operations. The aim of this
work was to compare MG method and standard meth-
ods namely the vanadomolybdate—VM method, and the
molybdenum blue—MB method, for accuracy, sensitiv-
ity and precision. The determined phosphorus contents
in oils were converted to equivalent of total PL levels.
2. Materials and methods
2.1. Materials
Samples at different stages of conventional technolo-
gical process: rape seeds (Brassica napus oleifera), rape
flakes from flakers, rape flakes from roaster ‘‘A’’, ‘‘B’’
‘‘C’’, pomace from oil presses (cake crushing), pressed
oil from cribriform storage, pressed oil after decanter,
extracted oil after last evaporator, extracted oil after
lecithin removal, and refined rapeseed oil were used.
2.2. Reagents
Ammonium phosphate (V) (5.0
105 M), ammonium
molybdate (VI) (0.1 M, 4.05
102 M, 5.30
103 M),
Malachite Green (MG) (2.0
103 M), polyvinyl alcohol
(PVA) (1%), ammonium metavanadate (V) (2.14
102
M), potassium dihydrogen ortophosphate (3.23
102,
3.23
103 M), hydrazine sulphate(VI) (3.07
103 M)
water solutions were prepared from analytical grade
reagents purchased from POCH, Gliwice, Poland. Stan-
dard solution of KH2PO4 (1 mg cm3) was obtained
from Merck, Germany.
2.3. Apparatus
Absorption spectra were recorded with a Helios
a-UNICAM spectrophotometer in a 1-cm quartz cell.
The hydrated gums from the oil were separated with a
lab centrifuge MLW K-70 (5000 rpm, 10 min).
2.4. Sample preparation
Samples of rape seeds, flakes, crude and refined oils
were digested as follows: 0.100 g of MgO and samples
(0.100–26.000 g) were weighed in quartz crucible, placed
in a furnace and ashed at 800  C. Blank digestions were
also carried out in the same way.
2.5. Analytical methods
2.5.1. Vanadomolybdate method (VM method)
5 cm3 of HNO3 (6.0 M), 10 cm3 of ammonium
molybdate (4.05
102 M), 10 cm3 of ammonium meta-
vanadate were added to ashed samples, and the absor-
bance was measured at 460 nm against a reagent blank,
after 20 min.
2.5.2. Molybdenum blue method (MB method)
The ashed samples were dissolved in 20 cm3 of redis-
tilled water containing 20 cm3 of 1 M H2SO4, and
transferred into 100-cm3 volumetric flask, followed by
the addition of ammonium molybdate (5.30
103 M)
and hydrazine sulphate (3.07
103 M) in 20 cm3 and
heated in boiling water bath for 20 min. After cooling,
the solution was made up to the mark and the absor-
bance measured at 730 nm against a reagent blank.
2.5.3. Malachite Green method (MG method)
The ashed samples were dissolved in 20 cm3 of redis-
tilled water containing sulphuric acid, transferred into
50-cm3 calibrated flask, and made up to the mark. Then
4 cm3 of the obtained solution, 6.8 cm3 of the ammo-
nium molybdate (0.1 M), 1.5 cm3 the Malachite Green
and 1 cm3 of the PVA solutions were placed in a 50-cm3
calibrated flask and made up to volume with water.
After 20 min, the absorbance of solution was measured
at 640 nm against a reagent blank.
The total phosphorus content in each sample was
determined (n=5 within 1 day) by the VM and MB
methods in order to compare with MG method. The
methods were compared for within day precision and
accuracy using the F-test and recovery study. The
 A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619 615

recovery experiments were performed by adding a fixed
amount of standard solution of KH2PO4 to the ana-
lysed samples.
2.6. Calibration curves
Calibration curves were obtained using working solu-
tions of KH2PO4 between 1.61
103–19.37
103 and
4.84
106–64.58
106 mol P dm3 for VM and MB
methods, respectively and (NH4)2HPO4 from
5.00
107–80.00
107 mol P dm3 for MG method.
Five calibration curves for each method were plotted on
the same day. The within day precision, known as
repeatability was found by regression analysis of
(A)=f(c) curves, and expressed as the relative standard
deviation (RSD%) of the slope (Miller & Miller, 2000).
The least-squares method was applied to calculate the
line of the best fit for the data (Table 1). The reported
MG method appeared to be more sensitive
(e=1.06
105 dm3 mol1 cm1) than standard methods
(VM method—e=28.57, MB method—e=1.19
104
dm3 mol1 cm1). The correlation coefficients were
0.9998 for MG method and 0.9999 for standard meth-
ods, values indicate the excellent precision of linear
calibration curves. The relative standard deviations
(RSD, n=5) of the curves slopes were 0.23, 0.36 and
0.45% for MG, VM and MB methods, respectively.
Precision of each method was tested by analysing five
replicate samples containing 3.00
106, 1.29
102 and
3.23
105 mol P dm3 for MG, VM and MB methods,
respectively. The values of RSD were less than 1%,
indicating reasonable intra-day (within day) repeat-
ability of the all three methods.
2.7. Influence of time on the absorbance variation
The optimum time required for the coloured complexes
formation using MG, VM and MB methods was found. In
the case of MG method, to reduce the colour development
time and improve the reproducibility of the absorbance,
PVA (stabilizing active agent) was used (Motomizu,
Wakimoto, & Toei, 1983). The absorbance measurements
were made every 20 min and results presented at Fig. 1. The
absorbance of MG-molybdophosphate ion-pair was con-
stant for the period 20–420 min, whereas of phosphovana-
domolybdate and molybdenum blue complexes varied

Table 1
Analytical parameters for the spectrophotometric determination of phosphorus

Parameters MG method Standard methods

VM method MB method

Concentration range [mol dm1] 5.00
107–8.00
106 1.61
103–1.94
102 4.84
106–6.46
105

Regression equation y=105497x +0.0009 y=28.38x +0.0012 y=11807x+0.0011
Relative standard deviation RSD of slopea [%] 0.23 0.36 0.45
Correlation coefficient (r) 0.9998 0.9999 0.9999
Relative standard deviation RSDb [%] 0.76 0.72 0.94
Mean molar absorptivity (E)a) [dm3/mol-cm] 1.06
105 28.57 1.19
104
a
n=5.
b
Five replicate samples containing 3.00
106, 1.29
102 and 3.23
105 mol Pdm3 for MG, VM and MB methods, respectively.

Fig. 1. Effect of time on absorbance of phosphorus solutions.

 616
Table 2
Determination of total phosphorus in studied samples

Statistical Samples

A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619

parameters
Rape seeds Rape flakes from Rape flakes from Rape flakes from Rape flakes from Pomace from Pressed oil Pressed oil after Extracted oil Extracted oil Refined
flakers roaster ‘‘A’’ roaster ‘‘B’’ roaster ‘‘C’’ oil presses from cribriform decanter after last after lecithin rapeseed
storage evaporator removal oil

MG method
Content Pa [mg/kg] 3758.91 6882.40 4937.80 5869.00 6530.40 7862.60 291.20 251.80 500.00 314.78 2.021
SDa [mg/kg] 13.05 38.89 17.12 24.87 29.11 27.97 1.30 2.28 3.61 1.91 0.014
RSDa [%] 0.35 0.57 0.35 0.42 0.45 0.36 0.45 0.91 0.72 0.61 0.69
Confidence limitb 3758.9116.21 6882.40 48.29 4937.8021.26 5869.00 30.88 6530.4036.14 7862.60 34.73 291.21.62 251.802.83 500.004.48 314.782.37 2.0210.017
[mg/kg]
VM method
Content Pa [mg/kg] 3751.41 6751.80 5063.40 5926.00 6559.60 7920.80 292.80 257.43 528.00 314.18 2.044
SDa [mg/kg] 19.24 34.32 31.29 20.60 26.26 32.91 2.77 3.08 2.74 1.94 0.016
RSDa [%] 0.51 0.51 0.62 0.35 0.40 0.42 0.95 1.20 0.52 0.62 0.78
Confidence limitb 3751.4123.89 6751.80 42.62 5063.4038.86 5926.00 25.58 6559.6032.61 7920.80 40.87 292.803.45 257.433.83 528.003.40 314.182.41 2.0440.020
[mg/kg]
MB method
Content Pa [mg/kg] 3743.90 7099.40 5550.60 5866.20 6594.00 7853.40 276.40 252.83 500.80 326.69 2.014
SDa [mg/kg] 24.40 24.34 39.07 43.28 17.73 48.87 2.97 1.31 6.42 3.76 0.016
RSDa [%] 0.65 0.34 0.70 0.74 0.27 0.62 1.07 0.52 1.28 1.15 0.79
Confidence limitb 3743.9030.30 7099.40 30.22 5550.6048.52 5866.20 53.74 6594.0022.02 7853.40 60.67 276.403.68 252.831.63 500.807.97 326.694.66 2.0140.020
[mg/kg]
a
n=5.
b
Probability level=0.95; SD—Standard deviation, RSD—Relative standard deviation.
 A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619 617

between 0.15–0.23 and 0.23–0.36, respectively in the same
time. Taking this into account, MG method was recom-
mended for the determination of total phosphorus content
in rape seeds and oils at various stages of refinement.
2.8. Calculation of total PL and NHP in rapeseed oils
dropwise. When water addition was completed, the
mixture was stirred at the same temperature for 15 min.
The hydrated gums from the hot oil were separated with
lab centrifuge MLW K-70 (5000 rpm, 10 min). Then the
top layer of oil was filtered and phosphorus determined
using MG, VM and MB methods.

Total phospholipids contents of the crude oils were

calculated basing of the elemental phosphorus contents. 3. Results and discussion
The ratio between PL content (%PL) and %P can be
expressed by the following equation: 3.1. Determination of total phosphorus

MWPL The total phosphorus content in rape seeds, flakes,

%PL ¼ %P ¼ %P26;
MWP
where: MWPL, MWP—molecular weights of phospholi-
pids and phosphorus, respectively; factor 26 was used
for conversion of % P to % PL (PN-88/A-86930).
NHP level was presented as the PL content in the fil-
tered oils after hydration.
2.7.1. Procedure of hydration of crude oils
Samples of oil (125 g) were heated to 80  C upon
stirring, than 50 cm3 of hot redistilled water was added
crude and refined oils at various stages of industrial
processes was determined using all three methods and
results listed in Table 2. The lowest concentrations of
phosphorus in refined oil were observed (2.021, 2.044,
2.014 mg P/kg for MG, VM and MB methods, respec-
tively). This fact indicated that the refining process of
crude vegetable oil ensures decrease of phosphorus
content in refined oil. The values of SD, RSD and con-
fidence limit were comparable to those obtained by VM
method and MB method. Therefore MG method can be

Table 3
Recovery tests

Sample Content Pa [mg/ml] (RSD [%]) Addedb P Found Pa [mg/ml]/Recovery [%] (RSD [%])
[mg/ml]
MG method VM method MB method MG method VM method MB method

A 0.4007 (0.45) 0.3999 (0.94) 0.3991 (1.58) 0.2000 0.6001 (0.63) 99.9 0.6009 (1.79) 100.2 0.5978 (0.98) 99.8
B 0.2958 (0.87) 0.3024 (1.41) 0.2970 (0.74) 0.1500 0.4460 (0.36) 100.0 0.4502 (0.67) 99.5 0.4497 (0.81) 100.6
C 0.2115 (0.31) 0.2111 (0.83) 0.2195 (0.77) 0.1000 0.3124 (0.57) 100.3 0.3165 (1.34) 101.7 0.3148 (1.29) 98.5
D 0.05192 (0.58) 0.05250 (1.45) 0.05174 (1.76) 0.0250 0.07624 (0.72) 99.1 0.07795 (0.95) 100.6 0.07743 (1.53) 100.9
a
n=5.
b
Standard solution of KH2PO4 (1 mg cm3), A—rape seeds (m=0.1066 g); B—crude oil after decanter (m=1.1747 g); C—crude oil after lecithin
removal (m=0.6719 g); D—refined rapeseed oil (m=25.6900 g).

Table 4
F-Snedecor test

Sample MG method Standard methods

Content Pa [mg/kg] s21 VM method MB method

a
Content P [mg/kg] s22 F1b Content Pa [mg/kg] s23 F2 b

Rape seeds 3758.91 170.34 3751.41 370.29 2.17 3743.90 595.30 3.49
Rape flakes from flakers 6882.40 1512.80 6751.80 1178.20 1.28 7099.40 592.30 2.55
Rape flakes from roaster ‘‘A’’ 4937.80 293.20 5063.40 979.30 3.34 5550.60 1526.80 5.21
Rape flakes from roaster ‘‘B’’ 5869.00 618.50 5926.00 424.50 1.46 5866.20 1873.20 3.03
Rape flakes from roaster ‘‘C’’ 6530.40 847.30 6559.60 689.80 1.23 6594.00 314.50 2.69
Pomace from oil presses 7862.60 782.30 7920.80 1083.20 1.38 7853.40 2387.80 3.05
Pressed oil from cribriform storage 291.20 1.70 292.80 7.70 4.53 276.40 8.80 5.18
Pressed oil after decanter 251.80 5.20 257.43 9.50 1.83 252.83 1.72 3.02
Extracted oil after last evaporator 500.00 13.00 528.00 7.50 1.73 500.80 41.20 3.17
Extracted oil after lecithin removal 314.78 3.64 314.18 3.76 1.03 326.69 14.11 3.87
Refined rapeseed oil 2.021 0.000192 2.044 0.000253 1.32 2.014 0.000254 1.32
a
n=5.
s2 s2
b
Probability level=0.95; s21, s22, s23—variance of results for MG, VM and MB methods, respectively; F1 ¼ s21 as s21 >s22 or F1 ¼ s22 as s22 >s21 and
s22 2 2 s23 2 2 2 1
F2 ¼ s2 as s2 >s3 or F2 ¼ s2 as s3 > s2
3 2
618 A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619

Removal
applicable to total phosphorus determinations in rape-

of PL

0.65830.0090 (1.09) 49.44

0.71920.0096 (1.07) 15.33

4.62

4.53
seed oil.

[%]
The validity of the proposed and standard methods

0.68540.0113 (1.33)

0.62760.0106 (1.35)
was evaluated. The results of recovery tests are listed in

NHPa confidence
Table 3. The recoveries of phosphorus added as

limitb [%] (RSD

KH2PO4 to real samples solutions were ranged between
99.1 and 100.3, 99.5–101.7, 98.5–100.9% for our assay,
VM and MB methods, respectively. Moreover repeat-
ability (calculated using RSD, n=5) for phosphorus
After hydration

determination by all three methods was below 2%.

Content Pa

Statistical analysis of the results obtained by MG,

[mg/kg]

263.60

253.20

276.60
241.4
VM and MB methods using the F-test (the variance
ratio F-test) revealed no significant difference between
MB method

the performed determinations of the applied methods at

hydration

of PL [%] PLa [%]

Before

the probability level 95%. The calculated F1 and F2

0.7186

0.6574

1.3021

0.8494
Removal Total

values (F1— the variance ratio of proposed MG and

VM method, F2- the variance ratio of proposed MG
and MB method) were collected in Table 4. The found
0.67340.0139 (1.63) 50.95

0.69470.0101 (1.17) 14.96

4.78

5.30

F1 and F2 values are less than the theoretical F=6.39.

The results presented in Tables 2–4 indicated that MG
0.72490.0150 (1.66)

0.63380.0170 (1.75)

method is accurate and precise.

Content Pa NHPa confidence
limitb [%] (RSD

3.2. Calculation of total PL and NHP in rapeseed oil

Because there are not simple and reliable methods for

routine analysis of PL level in edible oils, the total
After hydration

phosphorus contents were determined using the pro-

posed MG method and standard VM, MB methods and
PLa [%] [mg/kg]
Standard methods

278.80

244.80

255.00

267.20

then converted to equivalent PL levels.

The results of total PL and NHP calculations are
VM method

presented in Table 5. Repeatability (calculated using the

hydration

RSD, n=5) of all methods did not exceed 2%.

Removal Before

0.7613

0.6693

1.3728

0.8169
Total

The results listed in Table 5 indicated that, the hydra-

tion of crude oils caused 4.44–5.30% decrease of PL in
Determination of total and nonhydratable phospholipids in crude rapeseed oil

the pressed oils, 49.44–50.95% in the extracted oil after a

of PL

0.65100.0074 (0.92) 49.92

0.69680.0060 (0.70) 14.86

4.60

4.44
[%]

last evaporator and 14.86–15.33% in the extracted oil

after lecithin removal. The larger amounts of NHP was
0.62560.0049 (0.62)
0.72230.0098 (1.1)

detected in the pressed oils than in the extracted oils. The

NHPa confidence

Probability level=0.95; RSD—Relative standard deviation.

limitb [%] (RSD)

total PL and NHP contents determined by MG method

were comparable with those obtained by VM and MB
methods.
Factor 26 was used for conversion of P to PL values.
After hydration

4. Conclusion
Content Pa
[mg/kg]

277.80

240.60

250.40

268.00

The differences in phosphorus contents of samples

from several technological steps were observed. The
MG method

mean concentrations of phosphorus in rape seeds,

Total PLa
hydration

flakes, crude and refined oils determined by MG

Before

0.7571

0.6547

1.3000

0.8184
[%]

method agreed with those obtained by VM and MB

methods. Nevertheless, it seems to be more useful than
Extracted oil after

Extracted oil after

cribriform storage
Pressed oil from

standard procedures applied for total phosphorus deter-

lecithin removal
Pressed oil after

last evaporator

mination in vegetable oils so far. The advantage of the

n=5.

proposed MG method is the highest sensitivity

decanter
Table 5

Sample

(e=1.06
105, 28.57, 1.19
104 dm3 mol1 cm1 for MG,

b
a

VM and MB methods, respectively). The within-day

 A. Szyd•owska-Czerniak, E. Sz•yk / Food Chemistry 81 (2003) 613–619
precision expressed as the relative standard deviations
were less than 2% (n=5) and recovery which are close
to 100% for different samples, indicating reasonable
repeatability and accuracy of the all three methods. At
95% confidence level, the calculated F-test values did
not exceed the theoretical values, indicating that there is
no significant difference between the proposed MG
method and the standard VM and MB methods with
regard to accuracy and precision. The proposed MG
method is relatively simpler and convenient than stan-
dard methods. All three methods are inexpensive and
useful for determination of total phosphorus content in
oils at various stages of technological process, that can
be converted to equivalent PL value.
Acknowledgements
The authors wish to thank J.M. Rector of Nicholas
Copernicus University for the financial support grant
no. 508-Ch.
References
Altmann, H. J., Fürstenau, E., Gielewski, A., & Scholz, L. (1971).
Photometrische bestimmung kleiner phosphatmengen mit malachit-
grün. Fresenius Zeitschrift für Analytische Chemie, 256(4), 274–276.
Bosco, M., Culeddu, N., Toffanin, R., & Pollesello, P. (1997). Organic
solvent system for 31P nuclear magnetic resonance analysis of leci-
thin phospholipids: applications to two-dimensional gradient-
enhanced 1H-detected heteronuclear multiple quantum coherence
experiments. Analytical Biochemistry, 245(1), 38–47.
Carelli, A. A., Brevedan, M. I. V., & Crapiste, G. H. (1997). Quanti-
tative determination of phospholipids in sunflower oil. Journal of
the American Oil Chemists’ Society, 74(5), 511–514.
Cert, A., Moreda, W., & Pérez-Camino, M. C. (2000). Chromato-
graphic analysis of minor constituents in vegetable oils. Journal of
Chromatography A, 881(1-2), 131–148.
Chapman, G. W. Jr. (1980). A conversion factor to determine phos-
pholipid content in soybean and sunflower crude oils. Journal of the
American Oil Chemists’ Society, 57(9), 299–302.
Diehl, B. W. K. (2001). High resolution NMR spectroscopy. European
Journal of Lipid Science and Technology, 103(12), 830–834.
Dobson, G., & Deighton, N. (2001). Analysis of phospholipid mole-
cular species by liquid chromatography—atmospheric pressure che-
mical ionisation mass spectrometry of diacylglycerol nicotinates.
Chemistry and Physics of Lipids, 111(1), 1–17.
El-Sebaiy, L. A., El-Mahdy, A. R., Moustafa, E. K., & Mohamed,
M. S. (1980). A modified method for the isolation and quantifica-
tion of oil seed phospholipids. Food Chemistry, 5(3), 217–229.
619
Goh, S. H., Tong, S. L., & Gee, P. T. (1984). Total phospholipids in
crude palm oil: quantitative analysis and correlations with oil qual-
ity parameters. Journal of the American Oil Chemists’ Society,
61(10), 1597–1600.
Kanamoto, R., Wada, Y., Miyajima, G., & Kito, M. (1981). Phos-
pholipid-phospholipid interaction in soybean oil. Journal of the
American Oil Chemists’ Society, 58, 1050–1053.
Kang, D. H., & Row, K. H. (2001). High-purity separation of phos-
pholipids by preparative high-performance liquid chromatography.
Journal of Industrial and Engineering Chemistry, 7(3), 178–182.
Melton, S. L. (1992). Analysis of soybean lecithins and beef phospho-
lipids by HPLC with evaporative light scattering detector. Journal of
the American Oil Chemists’ Society, 69(8), 784–788.
Miller, J. N., & Miller, J. C. (2000). Statistic and chemometrics for
analytical chemistry (4th ed.). Harlow: Pearson Education.
Motomizu, S., Wakimoto, T., & Toei, K. (1983). Spectrophotometric
determination of phosphate in river waters with molybdate and
malachite green. Analyst, 108(1284), 361–367.
Mounts, T. L., Abidi, S. L., & Rennick, K. A. (1992). HPLC analysis
of phospholipids by evaporative laser light-scattering detection.
Journal of the American Oil Chemists’ Society, 69(5), 438–442.
Nomikos, T., Karantonis, H. C., Fragopoulou, E., & Demopoulos,
C. A. (2002). One-step separation system for the main phospholi-
pids, glycolipids, and phenolics by normal phase HPLC. Applica-
tion to polar lipid extracts from olive and sunflower oils. Journal of
Liquid Chromatography & Related Technologies, 25(1), 137–149.
Nzai, J. M., & Proctor, A. (1998). Phospholipids determination in
vegetable oil by thin-layer chromatography and imaging densito-
metry. Food Chemistry, 63(4), 571–576.
Ostrowska, J., Skrzydlewska, E., & Figaszewski, Z. A. (2000). Isola-
tion and analysis of phospholipids. Chemia Analityczna, 45(5), 613–
629.
Racicot, L. D., & Handel, A. P. (1983). Degumming of soybean oil:
quantitative analysis of phospholipids in crude and degummed oil.
Journal of the American Oil Chemists’ Society, 60(6), 1098–1101.
Rozentsvet, O. A., Dembitsky, V. M., & Saksonov, S. V. (2000).
Occurrence of diacylglyceryltrimethylhomoserines and major phos-
pholipids in some plants. Phytochemistry, 54(4), 401–407.
Singleton, J. A., Ruan, M., Sanford, J. H., Haney, C. A., & Stike-
leather, L. F. (1999). Separation and characterization of peanut
phospholipid molecular species using high-performance liquid
chromatography and fast atom bombardment mass spectrometry.
Journal of the American Oil Chemists’ Society, 76(1), 49–56.
Sotirhos, N., Herslöf, B., & Kenne, L. (1986). Quantitative analysis of
phospholipids by 31P-NMR. Journal of Lipid Research, 27(4), 386–
392.
Standard methods for the Analysis of Oils and Edible Vegetable Fats.
(1978). Determination de la teneur en phosphore (IUPAC 2.421).
Standard methods for the Analysis of Oils and Edible Vegetable Fats.
(1988). Determination of the phosphorus content (PN-88/A-86930).
Totani, Y., Pretorius, H. E., & du Plessis, L. M. (1982). Extraction of
phospholipids from plant oils and colorimetric determination of
total phosphorus. Journal of the American Oil Chemists’ Society,
59(4), 162–163.
Touchstone, J. C. (1995). Thin-layer chromatographic procedures for
lipid separation. Journal of Chromatography B, 671(1-2), 169–195.