AU2019101808A4 - Gas chromatography method for simultaneously detecting and separating multiple fatty acids - Google Patents
Gas chromatography method for simultaneously detecting and separating multiple fatty acids Download PDFInfo
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Abstract
The present invention discloses a gas chromatography method
capable of simultaneous detecting and separating of multiple fatty acids,
including: detecting by gas chromatography with a CP Sil 88 gas
chromatographic column using cyanopropyl siloxane as a stationary
phase, wherein a specification of the gas chromatographic column is
80-200 m x 0.25 mm x 0.20 pm, and detection conditions for the gas
chromatography include: an injection temperature: 200-230°C; an
injection volume: IpL; a split ratio: 8-12:1; a flow rate of nitrogen gas:
10-12 cm/s; a constant linear velocity mode; a detector: a
flame-ionization detector (FID), a detection temperature: 200-230°C, and
an end-puff volume: 2-5 mL/min. The present invention achieve
high-efficiency separation and qualitative and quantitative analysis of 72
fatty acids, and has the advantages of high throughput, high sensitivity,
and low limit of detection.
1
WO 2021/003792 PCT/CN2019/099735
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Description
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The present invention relates to the field of food detection, and in
particular to a gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids.
Fatty acids refer to a type of compounds composed of carbon,
hydrogen, and oxygen, and are also one of the main energy sources of
human body. Fatty acids contain short-chain fatty acids (C3-C5),
medium-chain fatty acids (C6-C12) and long-chain fatty acids (C13
C24), wherein, short-chain fatty acids are mainly derived from milk,
oligosaccharides, malted barley, oat bran, corn starch, etc., medium-chain
fatty acids are mainly derived from edible oils (especially coconut oil and
palm kernel oil), dairy and milk foods, etc, and long-chain fatty acids are
mainly derived from meat foods, edible oils and hydrogenated foods, etc.
The current detection methods of fatty acids in food mainly include
a gas chromatography method, a liquid chromatography method, a silver
ion thin layer chromatography method, an infrared spectroscopy method,
a mass spectrometry method, a capillary electrophoresis method, etc.;
wherein, the gas chromatography method is widely used due to its high separation efficiency and low limit of detection. However, With the rapid development of detection technology, It is urgent to establish a high-throughput, accurate identification and rapid detection technology for fatty acids, and the existing gas chromatography method for separation and detection of fatty acids need to be further improved.
Therefore, a new detection method which can achieve achieve
high-throughput, accurate qualitative and quantitative analysis of fatty
acids in food has good promotion and application value on the basis of
the existing gas chromatography method for separation and detection of
fatty acids.
An object of the present invention is to solve at least the above
problems, and to provide, at least, the advantages that will be described
later.
Another object of the present invention is to provide a gas
chromatography method capable of simultaneous detecting and
separating of multiple fatty acids, which can achieve high-efficiency
separation and qualitative and quantitative analysis of 72 fatty acids, and
has the advantages of high throughput, high sensitivity, and low limit of
detection.
To realize the objects mentioned above and other advantages, the
present invention provides a gas chromatography method capable of simultaneous detecting and separating of multiple fatty acids, including: detecting by gas chromatography with a CP Sil 88 gas chromatographic column using cyanopropyl siloxane as a stationary phase, wherein a specification of the gas chromatographic column is
-200 m x 0.25 mm x 0.20 pm, and detection conditions for the gas
chromatography include:
an injection temperature: 200-230°C; an injection volume: IpL; a
split ratio: 8-12:1; a flow rate of nitrogen gas: 10-12 cm/s; a constant
linear velocity mode;
keeping for 4-6 min at an initial temperature of 60-80°C, heating to
150-180°C at a heating rate of 20-30°C /min, keeping for 3-5 min at
150-180 0C, heating to 200-225 0C at a heating rate of 1-50 C/min, keeping
for 40-60 min at 200-225 0C, heating to 200-230C at a heating rate of
0.5-2 0C /min, keeping for 4-6 min at 200-230°C;
a detector: a flame-ionization detector (FID), a detection temperature:
200-230 0C, and an end-puff volume: 2-5 mL/min.
Preferably, the specification of the gas chromatographic column is
100 mx0.25 mmx0.20 pm.
Preferably, the detection conditions for gas chromatography include:
the injection temperature: 230°C; the injection volume: 1 L; the
split ratio: 10:1; the flow rate of nitrogen gas: 10.6 cm/s; the constant
linear velocity mode; keeping for 5 min at the initial temperature of 60°C, heating to
160°C at the heating rate of 25°C /min, keeping for 4 min at 160°C,
heating to 225°C at the heating rate of 2°C/min, keeping for 50 min at
225 0C, heating to 230 0C at the heating rate of 1°C /min, and keeping for
min at 230°C;
the detector: the flame-ionization detector (FID), the detection
temperature: 230 0C, and the end-puff volume: 3 mL/min.
Preferably, the multiple fatty acids include C3-C5 short-chain fatty
acids, C6-C12 medium-chain fatty acids, and C13- C24 long-chain fatty
acids.
Preferably, the multiple fatty acids include C3:O, C4:0, C5:0, C6:0,
C7:, C8:, C9:0, C1:, C:, C12:, Cll:l-lOc, C13:0, C12:1-11c,
C14:0, C13:1-12c, C14:1-9t, C14:1-9c, C15:0, C15:1-10t, C15:1-10c,
C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1-10t, C17:1-10c,
C18:, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c,
C19:0, C18:2-9t,12t, C19:1-7t, C19:1-10t, C19:1-7c, C19:1-10c,
C18:2-9c, 12c, C20:0, C20:1-11t, C18:3-6c, 9c, 12c, C20:1-5c, C20:1-8c,
C20:1-11c, C19:2-10c, 13c, C18:3-9c, 12c, 15c, C21:0, C18:2-9c, lit,
C18:2-10t, 12c, C21:1-12c, C20:2-11c, 14c, C22:0, C22:1-13t, C20:3-8c,
11c, 14c, C22:1-13c, C21:2-12c, 15c, C20:3-11c, 14c, 17c, C23:0,
C20:4-5c, 8c,I1c, 14c, C23:1-14c, C22:2-13c, 16c, C24:0, C20:5-5c, 8c,
11c, 14c, 17c, C24:1-15c, C22:3-13c, 16c, 19c, C22:4-7c, 10c, 13c, 16c,
C22:5-4c, 7c, 10c, 13c, 16c, C22:5-7c, 10c, 13c, 16c, 19c, C22:6-4c, 7c,
c, 13c, 16c, 19c.
Preferably, a limit of detection is 0.000084-0.001276 g/100 g, and a
limit of quantitation is 0.000289-0.004263 g/100 g.
Preferably, an RSD value of intra-day accuracy is controlled
between 0.57-9.81%, and an RSD value of inter-day accuracy is
controlled between 0.47-9.87%.
The present invention includes at least the following substantial
improvements and beneficial effects:
1. The present invention adopts a CP Sil 88 gas chromatographic
column using cyanopropyl siloxane as a stationary phase, and establishes
a high-throughput detection method capable of simultaneously detecting
72 kinds of C3-C24 fatty acids including 36 kinds of unsaturated fatty
acids, 22 kinds of saturated fatty acids, 12 kinds of trans fatty acids and 2
kinds of conjugated fatty acids. Compared with a detection method of the
national standard GB5009.168-2016 "Determination of fatty acids in
foods", the detection method of the present invention can detect more
than 35 kinds of fatty acids including 18 kinds of unsaturated fatty acids,
kinds of saturated fatty acids, 10 kinds of trans fatty acids and 2 kinds
of conjugated fatty acids.
2. The gas chromatography method of the present invention capable
of detecting 72 kinds of fatty acids, which has an RSD value of intra-day accuracy controlled between 0.57-9.81%, an RSD value of inter-day accuracy controlled between 0. 4 7 -9. 8 7 %, is lower than 10% required by the national standard GB 5009.168-2016, and is far lower than 15% required by the national standard GB 5009.257-2016. The gas chromatography method has good stability and meets the requirements of precise quantitative analysis.
3. The gas chromatography method of the present invention capable
of detecting 72 kinds of fatty acids has a limit of detection is
0.000084-0.001276 g/100 g, which shows that each fatty acid methyl
ester of 0.0013 g/100 g can be qualitatively detected under the conditions
of existing instruments and used parameters. The limit of detection is
two-tenths of a limit of detection (0.0013-0.0066 g/100 g) of the national
standard GB 5009.168-2016, and is one-tenth of a limit of detection
(0.012 g/100 g) of the national standard GB 5009.257-2016. A limit of
quantitation of the present invention is 0.000289-0.004263 g/100 g,
which shows that that each fatty acid methyl ester of 0.0043 g/100 g can
be quantitatively detected under the conditions of existing instruments
and used parameters. The limit of quantitation is two-tenths of a limit of
quantitation (0.024 g/100 g) of the national standard GB 5009.257-2016.
The detection method of the present invention can significantly reduce
the limit of detection and the limit of quantitation of fatty acids, has good
accuracy, and can achieve precise identification of fatty acids.
4. A linear relationship established in the gas chromatography
method capable of detecting 72 kinds of fatty acids of the present
invention has good linear correlation between an instrument response
value of each fatty acid and an concentration, and the linear relationship
is basically higher than 0.999, which fully meets the requirements of
quantitative analysis, and has wide adaptability.
5. The present invention can also realize high-efficiency separation
of 21 kinds of trans fatty acids, 3 kinds of cis-trans conjugated linoleic
acid and 2 kinds of trans-trans conjugated linoleic acid. Compared with a
detection method of the national standard GB5009.257-2016
"Determination of trans fatty acids in foods", the gas chromatography
method of the present invention can detect more than 12 kinds of fatty
acids including 7 kinds of trans fatty acids, 3 kinds of cis-trans
conjugated linoleic acid and 2 kinds of trans-trans conjugated linoleic
acid.
Other advantages, objects, and features of the present invention will
be shown in part through the following description, and in part will be
understood by those skilled in the art from study and practice of the
present invention.
Fig. 1 is a figure of temperature program of the gas chromatography
method according to the present invention.
Fig. 2 is a first gas chromatogram of 72 kinds of fatty acid methyl
esters of the present invention.
Fig. 3 is a second gas chromatogram of 72 kinds of fatty acid methyl
esters of the present invention.
Fig. 4 is a third gas chromatogram of 72 kinds of fatty acid methyl
esters of the present invention.
Fig. 5 is a gas chromatogram of 26 kinds of fatty acid isomers of the
present invention.
Fig. 6 is a gas chromatogram of peanut oil in Embodiment 1 of the
present invention.
Fig. 7 is a gas chromatogram of milk in Embodiment 2 of the present
invention.
Fig. 8 is a gas chromatogram of linseed oil in Embodiment 3 of the
present invention.
Fig. 9 is a gas chromatogram of milk in Comparative Example 1 of
the present invention.
Fig. 10 is a gas chromatogram of linseed oil in Comparative
Example 2 of the present invention.
The present invention will now be described in further detail
concerning the embodiments, to enable a person skilled in the field to
practice regarding the literal description of the specification.
It should be noted that terms such as "having", "including" and
"comprising" as used herein do not exclude presence or addition of one or
more other elements or combinations thereof.
It should be noted that the experimental methods described in the
following embodiments are all conventional methods unless otherwise
specified, and the reagents and materials are commercially available
unless otherwise specified.
A gas chromatography method capable of simultaneous detecting
and separating of multiple fatty acids, including: detecting by gas
chromatography with a CP Sil 88 gas chromatographic column using
cyanopropyl siloxane as a stationary phase, wherein a specification of the
gas chromatographic column is 80-200 m x 0.25 mm x 0.20 pm, and
detection conditions for gas chromatography are:
an injection temperature: 200-230°C; an injection volume: IpL; a
split ratio: 8-12:1; a flow rate of nitrogen gas: 10-12 cm/s; a constant
linear velocity mode;
keeping for 4-6 min at an initial temperature of 60-80°C, heating to
150-180°C at a heating rate of 20-30°C /min, keeping for 3-5 min at
150-180 0C, heating to 200-225 0C at a heating rate of 1-50 C/min, keeping
for 40-60 min at 200-225 0C, heating to 200-230C at a heating rate of
0.5-2 0C /min, keeping for 4-6 min at 200-230°C;
a detector: a flame-ionization detector (FID), a detection temperature:
200-230 0C, and an end-puff volume: 2-5 mL/min.
In another technical solution, the gas chromatographic column
specification is 100 m x 0.25 mm x 0.20pm.
In another technical solution, the detection conditions for gas
chromatography are:
the injection temperature: 230°C; the injection volume: 1 L; a split
ratio: 10:1; the flow rate of nitrogen gas: 10.6 cm/s; the constant linear
velocity mode.
As shown in Fig.1, an initial temperature of the gas chromatographic
column is 60 0 C, keeping for 5 min at the initial temperature, heating to
160 0C at the heating rate of 250 C /min, keeping for 4 min at 1600 C,
heating to 225 0 C at the heating rate of 2C/min, keeping for 50 min at
225 0C, heating to 230 0C at the heating rate of 1°C /min, and keeping for
min at 2300 C.
A detector: a flame-ionization detector (FID), a detection
temperature: 230 0C, and an end-puff volume: 3 mL/min.
In another technical solution, the multiple fatty acids comprise
C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and
C13- C24 long-chain fatty acids.
In another technical solution, the reference retention time of 72
kinds of fatty acid methyl esters are shown in Table 1, and the multiple
fatty acids comprise C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0,
Ci:0, C12:0, C11:1-1c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t,
C14:1-9c, C15:0, C15:1-10t, C15:1-10c, C16:0, C15:1-14c, C16:1-9t,
C16:1-9c, C17:0, C17:1-10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t,
C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19:0, C18:2-9t,12t,
C19:1-7t, C19:1-10t, C19:1-7c, C19:1-10c, C18:2-9c, 12c, C20:0,
C20:1-11t, C18:3-6c, 9c, 12c, C20:1-5c, C20:1-8c, C20:1-11c, C19:2-10c,
13c, C18:3-9c, 12c, 15c, C21:0, C18:2-9c, lit, C18:2-10t, 12c,
C21:1-12c, C20:2-11c, 14c, C22:0, C22:1-13t, C20:3-8c, 11c, 14c,
C22:1-13c, C21:2-12c, 15c, C20:3-11c, 14c, 17c, C23:0, C20:4-5c, 8c,
11c, 14c, C23:1-14c, C22:2-13c, 16c, C24:0, C20:5-5c, 8c, 11c, 14c, 17c,
C24:1-15c, C22:3-13c, 16c, 19c, C22:4-7c, 10c, 13c, 16c, C22:5-4c, 7c,
c, 13c, 16c, C22:5-7c, 10c, 13c, 16c, 19c, C22:6-4c, 7c, 10c, 13c, 16c,
19c.
The present invention adopts a CP Sil 88 gas chromatographic
column using cyanopropyl siloxane as a stationary phase, and establishes
a high-throughput detection method capable of simultaneously detecting
72 kinds of C3-C24 fatty acids including 36 kinds of unsaturated fatty
acids, 22 kinds of saturated fatty acids, 12 kinds of trans fatty acids and 2
kinds of conjugated fatty acids. Compared with a detection method of the
national standard GB5009.168-2016 "Determination of fatty acids in
foods", the detection method of the present invention can detect more
than 35 kinds of fatty acids including 18 kinds of unsaturated fatty acids
(C11:1-10c, C12:1-11c, C13:1-12c, C15:1-14c, C18:1-6c, C18:1-11c,
C19:1-7c, C19:1-10c, C20:1-5c, C20:1-8c, C19:2-10c,13c, C21:1-12c,
C21:2-12c,15c, C23:1-14c, C22:3-13c, 16c, 19c, C22:4-7c, 10c, 13c, 16c,
C22:5-4c, 7c, 10c, 13c, 16c and C22:5-7c, 10c, 13c, 16c, 19c), 5 kinds of
saturated fatty acids (C3:0, C5:0, C7:0, C9:0 and C19:0), 10 kinds of
trans fatty acids (C14:1-9t, C15:1-10t, C16:1-9t, C17:1-10t, C18:1-6t,
C18:1-11t, C19:1-7t,C19:1-10t,C20:1-11t and C22:1-13t) and 2 kinds of
conjugated fatty acids (C18:2-9c, I1t and C18:2-10t, 12c).
Table 1
reference retention No. name of fatty acid time (min)
1 C3:0 19.085
2 C4:0 19.958
3 C5:0 21.239
4 C6:0 22.455
5 C7:0 23.602
6 C8:0 24.733
7 C9:0 25.871
8 C10:0 27.103
9 C11:0 28.485
10 C12:0 30.059
11 C11:1-10c 30.139
12 C13:0 31.851
13 C12:1-11c 31.951
14 C14:0 33.87
C13:1-12c 33.995
16 C 14:1-9t 35.226
17 C 14:1-9c 35.932
18 C15:0 36.097
19 C15:1-10t 37.585
C15:1-10c 38.335
21 C16:0 38.502
22 C1I5:1-14c 38.658
23 C1I6:1-9t 39.873
24 C1I6:1-9c 40.522
C17:0 41.034
26 C17:1-10t 42.474
27 C17:1-10c 43.132
28 C18:0 43.67
29 C1I8:1-6t 44.877
C1I8:1-9t 44.995
31 C18:1-llt 45.137
32 C1I8:1-6c 45.44
33 C1I8:1-9c 45.593
34 C18:i-iic 45.835
C19:0 46.394
36 C18:2-9t, 12t 47.075
37 C1I9:1-7t 47.663
38 C19:i-10t 47.808
39 C1I9:1-7c 48.198
C19:i-i0C 48.429
41 C18:2-9c, 12c 48.501
42 C20:0 49.277
43 C20:i-iit/Cl8:3-6c, 9c, 12c/C20:1-Sc 50.804
44 C20:i-8c 51.217
C20:i-iic 51.461
46 C19:2-10c, 13c 51.565
47 C18:3-9c, 12c, 15c 52.117
48 C21:0 52.375
49 C18:2-9c, lit 52.527
C 18:2-10t,12c 53.041
51 C21:i-12c 54.723
52 C20:2-1Iic, 14c 54.834
53 C22:0 55.741
54 C22:i-13t/C20:3-8c,li1c, 14c 57.58
C22:i-13c 58.345
56 C21:2-12c, 15c 58.471
57 C20:3-11c, 14c, 17c 59.151
58 C23:0 59.534
59 C20:4-5c, 8c, 11c, 14c 59.915
60 C23:1-14c/C22:2-13c, 16c 62.374
61 C24:0 63.674
62 C20:5-5c, 8c, 11c, 14c, 17c 65.197
63 C24:1-15c 66.961
64 C22:3-13c, 16c, 19c 67.884
65 C22:4-7c, 10c, 13c, 16c 69.587
66 C22:5-4c, 7c, 10c, 13c, 16c 72.375
67 C22:5-7c, 10c, 13c, 16c, 19c 76.62
68 C22:6-4c, 7c, 10c, 13c, 16c, 19c 80.147
The precision of an instrument is indicated by the relative standard
deviation (RSD), which is used to evaluate the reproducibility of a
measurement result of the same sample by the gas chromatography. The
experiment of the present invention adopts the same gas chromatography
method to analyse the intra-day (7 times a day) accuracy and the inter-day
(determination for 7 consecutive days) accuracy of a mixed standard
solution of 72 kinds of fatty acids, and the results of the intra-day
accuracy and the inter-day accuracy of 72 kinds of fatty acids are shown
as Table 2, wherein an RSD value of intra-day accuracy is controlled between 0.57-9.81%, and an RSD value of inter-day accuracy is controlled between 0.47-9.87%, which is lower than 10% required by the national standard GB 5009.168-2016, and is far lower than 15% required by the national standard GB 5009.257-2016. The detection method has good stability and meets the requirements of precise quantitative analysis.
Table 2
intra-day accuracy inter-day accuracy
confide confide
nce nce
limit limit name of No. averag (95 % averag (95% fatty acid RSD RSD e confide e confide
nce nce
interval interval
34497- 34450 1 C3:0 34679 0.57% 34784 1.04% 34860 35119
45259- 44569 2 C4:0 45550 0.69% 44990 1.01% 45841 45412
59925- 63376 3 C5:0 63726 6.45% 66592 5.22% 67526 69807
4 C6:0 65643 61485- 6.85% 63172 61488- 2.88%
69799 64856
58323- 66923 C7:0 59394 1.95% 73270 9.37% 60465 79618
65105- 75569 6 C8:0 66233 1.84% 80026 6.02% 67361 84482
65099- 74245 7 C9:0 66354 2.04% 79866 7.61% 67608 85486
69058- 79348 8 C1O:0 70399 2.06% 85872 8.21% 71740 92395
70885- 77878 9 C11:0 72359 2.20% 85030 9.09% 73832 92181
73956- 87760 C12:0 75111 1.66% 93175 6.28% 76266 98589
68439- 86559 11 C11:1-10c 70599 3.31% 92354 6.78% 72758 98149
72629- 84852 12 C13:0 74052 2.08% 91003 7.31% 75475 97153
71665- 86951 13 C12:1-11c 73382 2.53% 94378 8.51% 75099 101804
73295- 83196 14 C14:0 74242 1.38% 91556 9.87% 75189 99917
C13:1-12c 74612 72916- 2.46% 92339 84078- 9.67%
76307 100599
75196- 85126 16 C14:1-9t 76844 2.32% 93393 9.57% 78491 101660
61212- 71158-8 17 C14:1-9c 62294 1.88% 78012 9.50% 63375 4866
72108- 79252 18 C15:0 73176 1.58% 86187 8.70% 74244 93123
72508- 77291 19 C15:1-10t 73625 1.64°o 83923 8.55°o 74741 90556
69982- 84186 C15:1-10c 71409 2.16% 92044 9.23% 72835 99901
43993- 60972 21 C16:0 48385 9.81°o 64436 5.81°o 52775 67900
68273- 72414 22 C15:1-14c 69730 2.26°o 78146 7.93°o 71185 83877
75437- 80470 23 C16:1-9t 76583 1.62% 87578 8.77% 77728 94685
70567- 78332 24 C16:1-9c 71598 1.56% 85363 8.91% 72629 92394
30874- 31531 C17:0 31403 1.82% 32846 4.33% 31930 34161
26 C17:1-10t 69666 68424- 1.93% 79518 72707- 9.26%
70907 86330
68163- 72461 27 C17:1-10c 69380 1.90% 79278 9.30% 70597 86095
53040- 63326 28 C18:0 54369 2.64% 68807 8.61% 55696 74287
68599- 72832 29 C18:1-6t 70149 2.39% 80045 9.74% 71698 87258
71381- 75369 C18:1-9t 73069 2.50% 82148 8.92% 74756 88927
69985- 75497 31 C18:1-11t 71574 2.40% 81045 7.40% 73162 86592
70916- 78216 32 C18:1-6c 72632 2.55% 85493 9.20% 74347 92770
150091 78381- 15309 33 C18:1-9c 82817 5.79% -15609 2.12% 87252 4 6
70549- 75447 34 C18:1-11c 71966 2.13% 80956 7.36% 73382 86465
56191- 76965 C19:0 57603 2.65% 83023 7.89% 59013 89081
C18:2-9t, 68718- 72923 36 70191 2.27% 79583 9.05% 12t 71663 86243
67002- 71446 37 C19:1-7t 68750 2.75% 78088 9.20% 70498 84730
69103- 75474 38 C19:1-10t 70867 2.69% 82968 9.77% 72631 90461
66884- 77170 39 C19:1-7c 68756 2.94% 83616 8.34% 70626 90062
144954 202283 15440 21339 C19:1-10c -16385 6.62% -22450 5.63% 3 4 1 5
C18:2-9c, 14805- 17262 41 15192 2.75% 18065 4.80% 12c 15578 18868
2489-2 2538-2 42 C20:0 2690 8.05% 2673 5.43% 889 807
179558 197466 18377 21572 43 C20:1-11t -18798 2.48% -23398 9.15% 2 7 6 7
145084 147903 C18:3-6c, 14609 14812 44 -14710 0.69% -14835 1.52% 9c,12c 2 9 0 4
75183- 75693 C20:1-5c 75264 1.07% 75842 1.97% 75345 75991
46 C20:1-8c 65335 63628- 2.82% 78339 72086- 8.63%
67041 84592
102259 78775- 11127 47 C20:1-11c 81115 3.12% -12029 8.76% 83454 9 9
C19:2-10c, 56394- 59801 48 57987 2.97% 64313 7.58% 13c 59580 68824
C18:3-9c, 82884- 82564 49 84512 2.08% 82937 0.49% 12c,15c 86140 83310
80378- 85815 C21:0 82238 2.44% 92621 7.95% 84097 99428
C18:2-9c, 65162- 73591 51 66836 2.71% 80045 8.72% lit 68508 86500
C18:2-10t, 69840- 82368 52 71656 2.74% 89700 8.84% 12c 73471 97033
74272- 78168 53 C21:1-12c 76327 2.91% 85927 9.76% 78381 93687
C20:2-11c, 54809- 60159 54 57604 5.25% 61677 2.66% 14c 60398 63195
20604- 23889 C22:0 21202 3.05% 25725 7.72% 21799 27561
53884- 58446 56 C22:1-13t 57177 6.23% 62867 7.60% 60470 67288
C20:3-8c, 78545- 79511-7 57 79298 0.95% 79678 2.10% 11C, 14c 80051 9845
75451- 83341 58 C22:1-13c 77168 2.40% 91105 9.21% 78884 98868
C21:2-12c, 49899- 64612 59 54414 8.97% 67468 4.58% 15c 58928 70324
C20:3-11c, 63380- 78205 64904 2.54% 83667 7.06% 14c,17c 66428 89129
116180 155344 11917 16860 61 C23:0 -12216 2.72% -18186 8.50% 4 4 7 4
C20:4-5c, 65104- 79691 62 66838 2.80% 85210 7.00% 8c,lic,14c 68571 90729
74697- 81274 63 C23:1-14c 76288 2.26% 84507 4.14% 77879 87740
C22:2-13c, 41556- 41603 64 41608 1.26% 41710 2.57% 16c 41660 41818
32934- 41764 C24:0 34743 5.63% 44347 6.30% 36552 46929
C20:5-5c, 17952- 21292 66 8c, 11c, 14c, 18495 3.17% 23237 9.05% 19037 25182 17c
62537- 92637 67 C24:1-15c 63962 2.41% 94727 2.39% 65387 96818
C22:3-13c, 24887- 32815 68 25518 2.67% 34723 5.94% 16c,19c 26147 36632
C22:4-7c, 25895- 36171 69 26530 2.59% 36814 1.89% 10c, 13c,16c 27164 37456
C22:5-4c, 25882- 36093 7c, 10c, 13c, 26512 2.57% 36252 0.47% 27141 36411 16c
C22:5-7c, 20023- 31429 71 10c, 13c, 20603 3.04% 33294 6.05% 21183 35158 16c,19c
C22:6-4c, 23010- 31861 72 7c, 10c, 13c, 23463 3.04% 33518 5.34% 23916 35175 16c,19c
The limit of detection and the limit of quantitation of the 72 kinds of
fatty acids are shown as Table 3, which shows that the limit of detection
of the present invention is 0.000084-0.001276 g/100 g. Therefore, each
fatty acid methyl ester of 0.0013 g/100 g can be qualitatively detected
under the conditions of existing instruments and used parameters. The
limit of detection is two-tenths of a limit of detection (0.0013-0.0066
g/100 g) of the national standard GB 5009.168-2016, and is one-tenth of a limit of detection (0.012 g/100 g) of the national standard GB
5009.257-2016. The limit of quantitation of the present invention is
0.000289-0.004263 g/100 g, which shows that that each fatty acid methyl
ester of 0.0043 g/100 g can be quantitatively detected under the
conditions of existing instruments and used parameters. The limit of
quantitation is two-tenths of a limit of quantitation (0.024 g/100 g) of the
national standard GB 5009.257-2016. The detection method of the
present invention can significantly reduce the limit of detection (LOD)
and the limit of quantitation (LOQ) of fatty acids, has good accuracy, and
can achieve precise identification of fatty acids.
Table 3
NO. name of fatty acid LOD (g/100 g) LOQ (g/100 g)
1 C3:0 0.000542 0.001807
2 C4:0 0.000415 0.001385
3 C5:0 0.000310 0.001032
4 C6:0 0.000199 0.000663
5 C7:0 0.000154 0.000513
6 C8:0 0.000142 0.000474
7 C9:0 0.000141 0.000468
8 C10:0 0.000135 0.000451
9 C11:0 0.000136 0.000452
10 C12:0 0.000136 0.000454
11 C11:1-10c 0.000143 0.000475
12 C13:0 0.000143 0.000476
13 C12:1-11c 0.000145 0.000484
14 C14:0 0.000147 0.000490
C13:1-12c 0.000149 0.000495
16 C14:1-9t 0.000148 0.000492
17 C14:1-9c 0.000165 0.000550
18 C15:0 0.000155 0.000518
19 C15:1-10t 0.000159 0.000530
C15:1-10c 0.000164 0.000548
21 C16:0 0.000291 0.000969
22 C15:1-14c 0.000169 0.000565
23 C16:1-9t 0.000156 0.000520
24 C16:1-9c 0.000168 0.000561
C17:0 0.000377 0.001255
26 C17:1-10t 0.000174 0.000581
27 C17:1-10c 0.000176 0.000587
28 C18:0 0.000232 0.000773
29 C18:1-6t 0.000191 0.000635
C18:1-9t 0.000172 0.000573
31 C18:1-1lt 0.000181 0.000603
32 C18:1-6c 0.000176 0.000588
33 C18:1-9c 0.000177 0.000591
34 C18:1-11c 0.000177 0.000591
C19:0 0.000221 0.000737
36 C18:2-9t,12t 0.000185 0.000617
37 C19:1-7t 0.000204 0.000681
38 C19:1-lOt 0.000188 0.000626
39 C19:1-7c 0.000193 0.000642
C19:1-10c 0.000180 0.000599
41 C18:2-9c, 12c 0.000188 0.000628
42 C20:0 0.000886 0.002952
43 C20:1-1lt 0.000087 0.000293
44 C18:3-6c, 9c, 12c 0.000551 0.000187
C20:1-5c 0.000420 0.000155
46 C20:1-8c 0.000214 0.000714
47 C20:1-11c 0.000195 0.000651
48 C19:2-10c, 13c 0.000214 0.000712
49 C18:3-9c, 12c, 15c 0.000186 0.000621
C21:0 0.000458 0.001525
51 C18:2-9c, lit 0.000515 0.001716
52 C18:2-1t, 12c 0.000636 0.002119
53 C21:1-12c 0.000228 0.000760
54 C20:2-11c, 14c 0.000228 0.000758
55 C22:0 0.000228 0.000761
56 C22:1-13t 0.000802 0.002674
57 C20:3-8c, 11c, 14c 0.000425 0.002500
58 C22:1-13c 0.000259 0.000864
59 C21:2-12c, 15c 0.000263 0.000875
60 C20:3-11c, 14c, 17c 0.000272 0.000907
61 C23:0 0.000182 0.000606
62 C20:4-5c, 8c, 11c, 14c 0.000270 0.000901
63 C23:1-14c 0.000299 0.000998
64 C22:2-13c, 16c 0.000728 0.002443
65 C24:0 0.000663 0.002211
66 C20:5-5c, 8c, 11c, 14c, 17c 0.001102 0.003675
67 C24:1-15c 0.000338 0.001127
68 C22:3-13c, 16c, 19c 0.000840 0.002799
69 C22:4-7c, 10c, 13c, 16c 0.000840 0.002800
70 C22:5-4c, 7c, 10c, 13c, 16c 0.000889 0.002964
71 C22:5-7c, 10c, 13c, 16c, 19c 0.001280 0.004265
C22:6-4c, 7c, 10c, 13c, 16c, 72 19c 0.001219 0.004062
The standard curve, correlation coefficient and concentration range
of 72 kinds of fatty acids are shown in Table 4. The standard solutions of
72 kinds of fatty acid methyl ester are performed gradient dilution, the diluted mixed standard solutions of each concentration are analyzed by the gas chromatography according to an established method, and linear fitting is performed within a detected concentration range to obtain linear equations and correlation coefficients of each fatty acid methyl ester. the linear relationship is basically higher than 0.999, which fully meets the requirements of quantitative analysis.
Table 4
correlation name of fatty standard curve concentration No. coefficient acid (mg/L) range (mg/L) R2
1 C3:0 y= 515.72x + 638.05 0.9991 1.905-243.9
2 C4:0 y= 744.37x + 2072.3 0.9991 1.905-243.9
3 C5:0 y= 1012.9x + 1139.2 0.9999 1.905-243.9
4 C6:0 y= 1077.5x + 482.28 0.9998 1.905-243.9
C7:0 y= 1202.7x + 323.39 0.9999 1.905-243.9
6 C8:0 y= 1232.7x + 592.66 0.9999 1.905-243.9
7 C9:0 y= 1230.7x + 909.79 0.9999 1.905-243.9
8 C1O:0 y= 1298.7x + 931.85 0.9999 1.905-243.9
9 C11:0 y = 1322.1x + 1179 0.9999 1.905-243.9
C12:0 y= 1375.9x + 796.79 0.9998 1.905-243.9
11 C11:1-10c y= 1268.2x + 1871.7 0.9998 1.905-243.9
12 C13:0 y = 1346x + 1254.6 0.9997 1.905-243.9
13 C12:1-11c y= 1322.2x + 1254.4 0.9999 1.905-243.9
14 C14:0 y= 1342.2x + 1299.1 0.9991 1.905-243.9
C13:1-12c y= 1322.5x + 2110.2 0.9998 1.905-243.9
16 C14:1-9t y = 1359.2x + 1593 0.9997 1.905-243.9
17 C14:1-9c y= 1226.1x + 1930.3 0.9998 1.715-219.5
18 C15:0 y= 1292.8x + 2062.2 0.9998 1.905-243.9
19 C15:1-10t y= 1303.6x + 1925.1 0.9998 1.905-243.9
C15:1-10c y = 1272.2x + 1282 0.9996 1.905-243.9
21 C16:0 y= 696.36x + 1974.7 0.9997 1.905-243.9
22 C15:1-14c y= 1235.1x + 2298.9 0.9998 1.905-243.9
23 C16:1-9t y= 1356.8x + 2220.8 0.9997 1.905-243.9
24 C16:1-9c y= 1272.4x + 1043.8 0.9995 1.905-243.9
C17:0 y= 552.22x + 2023.4 0.9994 1.905-243.9
26 C17:1-10t y= 1243.6x + 2738.2 0.9994 1.905-243.9
27 C17:1-10c y = 1238.5x + 2076 0.9994 1.905-243.9
28 C18:0 y = 949.82x + 1977 0.9996 1.905-243.9
29 C18:1-6t y= 1324.9x + 499.24 0.9996 1.905-243.9
C18:1-9t y= 1257.7x + 3139.3 0.9993 1.905-243.9
31 C18:1-11t y= 1235.7x + 2933.8 0.9997 1.905-243.9
32 C18:1-6c y = 1314.3x + 1994 0.9998 1.905-243.9
33 C18:1-9c y = 1263x + 1760.6 0.9998 1.905-243.9
34 C18:1-11c y = 1290x + 1409.5 0.9998 1.905-243.9
C19:0 y= 1043.6x + 2051.1 0.9999 1.905-243.9
36 C18:2-9t,12t y= 1267.6x + 1873.7 0.9998 1.905-243.9
37 C19:1-7t y= 1277.2x + 1191.3 0.9998 1.905-243.9
38 C19:1-10t y= 1255.2x + 2649.2 0.9995 1.905-243.9
39 C19:1-7c y= 1248.1x + 1797.6 0.9998 1.905-243.9
C19:1-10c y= 1300.7x + 1741.7 0.9993 3.81-60.98
41 C18:2-9c, 12c y= 1175.7x + 1110.5 0.9918 1.905-60.98
42 C20:0 y= 268.77x + 561.73 0.9999 7.622-243.9
43 C20:1-11t y= 8422.8x + 1422.5 0.9996 0.762-97.6
C18:3-6c, 9c, 44 y= 3369.1x + 1422.5 0.9996 1.905-243.9 12c
C20:1-5c y= 3369.1x + 1422.5 0.9996 1.905-243.9
46 C20:1-8c y= 3369.1x + 1422.5 0.9996 1.905-243.9
47 C20:1-11c y= 3369.1x + 1422.5 0.9996 1.905-243.9
48 C19:2-1Oc, 13c y= 1202.4x + 2128.6 0.9998 1.905-243.9
C18:3-9c, 12c, 49 y = 1506.3x - 1143.5 0.9954 1.905-121.95 15c
C21:0 y= 1074.2x + 4344.3 0.997 1.905-121.95
51 C18:2-9c, 1lt y= 1444.7x + 1164.2 0.9995 1.905-243.9
52 C18:2-10t, 12c y= 610.87x + 43.034 0.9994 1.905-243.9
53 C21:1-12c y = 577.5x + 1517.5 0.9984 1.905-243.9
54 C20:2-11c, 14c y= 1254.5x + 2139.7 0.9998 1.905-243.9
C22:0 y = 1660.6x - 7610.1 0.9921 1.905-243.9
56 C22:1-13t y= 1096.1x + 2787.2 0.9959 3.811-121.95
C20:3-8c, lc, 57 y= 5235.2x + 1045.9 0.9998 0.381-48.78 14c
58 C22:1-13c y = 365.44x + 160.1 0.9999 3.81-243.9
59 C21:2-12c, 15c y= 5235.2x + 1045.9 0.9998 0.381-48.78
C20:3-11c, y = 1047x + 1045.9 0.9998 1.905-243.9 14c, 17c
61 C23:0 y = 1468.7x - 3678 0.9977 1.905-243.9
C20:4-5c, 8c, 62 y= 962.62x + 5942.1 0.9957 1.905-243.9 11c, 14c
63 C23:1-14c y= 1190.1x + 1762.5 0.9998 1.905-243.9
64 C22:2-13c,16c y= 7283.1x + 388.67 0.9998 1.905-243.9
C24:0 y= 2040.4x + 1081.7 0.9999 1.905-243.9
C20:5-5c,8c,11 66 y= 1224.5x + 1525.6 0.9998 1.905-243.9 c,14c,17c
67 C24:1-15c y= 7283.1x + 388.67 0.9998 1.905-243.9
C22:3-13c,16c, 68 y= 1456.6x + 388.67 0.9998 0.381-48.78 19C
C22:4-7c,10c,1 69 y = 554.25x + 685.4 0.9991 1.905-243.9 3c,16c
C22:5-4c, 7c, y= 1694.6x + 730.83 0.9998 0.762-48.78 lOc,13c,16c
C22:5-7c, 10c, 71 y= 1263.6x - 340.87 0.9996 1.905-243.9 13c,16c,19c
C22:6-4c,7 c,
72 10c, 13c, 16c, y=2451.7x+282.75 0.9998 0.381-48.78
19c
The presenti invention can also realize high-efficiency separation of
21 kinds of trans fatty acids, 3 kinds of cis-trans conjugated linoleic acid
and 2 kinds of trans-trans conjugated linoleic acid. As shown in Fig. 5,
each peak is numbered sequentially in Arabic numerals, and corresponds
to each fatty acid in Table 5, wherein, an unmarked peak between No. 11
and No. 12 is the peak of C18: 2-9c, 12c (corresponding to No. 41 in the
Table 1), and unmarked peak between No. 17 and No. 18 is the peak of
C18: 3-9c, 12c, 15c (corresponding to No. 49 in the Table 1). The two
compounds are not numbered in Fig. 5 in order not to affect the display of
the 26 kinds fatty acid isomers. Compared with a detection method of the
national standard GB5009.257-2016 "Determination of trans fatty acids
in foods", the present invention can detect more than 12 kinds of fatty
acids including 7 kinds of trans fatty acids (C14:1-9t, C15:1-10t,
C17:1-10t, C19:1-7t, C19:1-10t, C18:2-9c, 12t, C18:2-9t, 12c), 3 kinds of
cis-trans conjugated linoleic acid (C18:2-9c, 1lt, C18:2-10t, 12c and
C18:2-11c, 13t) and 2 kinds of trans-trans conjugated linoleic acid
(C18:2-9t, 1lt and C18:2-10t, 12t). The reference retention time of 26
kinds of fatty acid isomers are shown in Table 5.
Table 5
reference retention No. name of fatty acids time (min)
1 C14:1-9t 35.09
2 C15:1-10t 37.45
3 C16:1-9t 39.736
4 C17:1-10t 42.341
5 C18:1-6t 44.751
6 C18:1-9t 44.867
7 C18:1-11t 45.009
8 C18:2-9t, 12t 46.937
9 C19:1-7t 47.483
10 C18:2-9c, 12t/C19:1-10t 47.684
11 C18:2-9t, 12c 47.866
12 C18:3-9t, 12t, 15t 49.394
13 C18:3-9t, 12t, 15c/C18:3-9t, 12c, 15t 50.242
14 C18:3-9c, 12t, 15t/C20:1-11t 50.471
15 C18:3-9c, 12c, 15t 50.671
16 C18:3-9c, 12t, 15c 51.28
17 C18:3-9t, 12c, 15c 51.38
18 C18:2-9c, lit 52.302
19 C18:2-11c, 13t 52.742
20 C18:2-10t, 12c 52.884
21 C18:2-9t, 1lt/C18:2-1t, 12t 53.949
22 C22:1-13t 57.215
Embodiment 1
A qualitative and quantitative analysis of fatty acids in peanut oil by
the gas chromatography method, includes:
weighing 100 mg of peanut oil in a 10 mL centrifuge tube, adding 2
mL of an C11:0 internal standard solution, adding 0.1 mL of a 2 mol/L of
a methanol solution of potassium hydroxide, performing vortex mixing
for 30 s, centrifuging at 4000 rpm for 10 min, diluting 20 pL of the
supernatant in a 1 mL volumetric flask to 1 mL, and detecting by
Shimadzu GC-2010 gas chromatography. A detection condition for the
gas chromatography are: gas chromatography column: CP Sil 88
(100mx0.25mmx0.20pm), an injection temperature: 200°C, an injection
volume: 1 L, a split ratio: 8:1, a flow rate: 10 cm/s (nitrogen gas); a
constant linear velocity mode. A temperature program of the gas
chromatography column box is: keeping for 5 min at 60°C, heating to
150°C at a heating rate of 21°C /min, keeping for 5 min at 150°C, heating
to 200°C at a heating rate of1C/min, keeping for 60 min at 200°C,
heating to 200°C at a heating rate of 0.5°C /min, and keeping for 6 min at
200°C. A detector is flame-ionization detector (FID), detection
temperature is 200°C, and an end-puff volume is 2.0 mL/min.
As shown in Fig. 6, the peanut oil detected by the above detection
method contains 10 fatty acids, wherein the contents of oleic acid
(C18:1-9c), linoleic acid (C18:2-9c, 12c) and palmitic acid (C16:0) are
higher, and alsoinclude stearic acid (C18:0), C18:1-11c, C20:0,
C20:1-11c, C18:3-9c, 12c, 15c, C22:0 and C22:1- 13c.
Embodiment 2
A qualitative and quantitative analysis of fatty acids in milk by the
gas chromatography method, includes:
weighing 1 mL of milk in a flask, adding 5 mL of ammonia water,
mixing, hydrolyzing in a 70-80°C water bath for 20 min, shaking the
flask every 5 minutes to mix particles adhered to a wall of the flask into a
solution, cooling to room temperature after hydrolysis, adding 10 mL of
% ethanol, mixing, transferring the solution in the flask to a separating
funnel, rinsing the flask and a stopper of the flask with 50 mL of a mixed
solution of ether and petroleum ether, merging into the separating funnel,
covering, shaking for 5 min, standing for 10 min, collecting the ether
layer extract into a 250 mL flask, repeating extraction of the solution for
3 times according to the above steps, rinsing the separating funnel with
the mixed solution of ether and petroleum ether, collecting in a 250 mL
flask, concentrating with a rotary evaporator to obtain fat extract, adding
2 mL of an C11:0 internal standard solution into the fat extract, adding 1
mL of isooctane and 0.1 mL of a 2 mol/L of a methanol solution of
potassium hydroxide, performing vortex mixing for 30 s, centrifuging at
4000 rpm for 10 min, diluting 20 pL of the supernatant in a 1 mL
volumetric flask to 1 mL, and detecting by Shimadzu GC-2010 gas
chromatography. A detection condition for the gas chromatography are:
gas chromatography column: CP Sil 88 (100mx0.25mmx0.20pm),
injection temperature: 230°C, an injection volume: 1 L, a split ratio:
11.5:1, a flow rate: 11.6 cm/s (nitrogen gas); a constant linear velocity
mode. A temperature program of a gas chromatography column box is:
keeping for 4 min at 80°C, heating to 180°C at a heating rate of 29°C
/min, keeping for 3 min at 180°C, heating to 225°C at a heating rate of
4.5°C/min, keeping for 40 min at 225°C, heating to 230°C at a heating
rate of 2°C /min, and keeping for 4 min at 230°C. A detector is
flame-ionization detector (FID), detection temperature is 230°C, and an
end-puff volume is 3.8 mL/min.
As shown in Fig. 7, the milk detected by the above detection method
contains 10 fatty acids, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0,
palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1-9c) and
linoleic acid (C18:2-9c, 12c).
Embodiment 3
A qualitative and quantitative analysis of fatty acids in linseed oil by the gas chromatography method, includes: weighing 100 mg of linseed oil in a 10 mL centrifuge tube, adding 2 mL of an C11:0 internal standard solution, adding 0.1 mL of a 2 mol/L of a methanol solution of potassium hydroxide, performing vortex mixing for 30 s, centrifuging at 4000 rpm for 10 min, diluting 20 pL of the supernatant in a 1 mL volumetric flask to 1 mL, and detecting by
Shimadzu GC-2010 gas chromatography. A detection condition for the
gas chromatography are: gas chromatography column: CP Sil 88
(100mx0.25mmx0.20pm), injection temperature: 230°C, an injection
volume: 1 L, a split ratio: 10:1, a flow rate: 10.6 cm/s (nitrogen gas); a
constant linear velocity mode. A temperature program of a gas
chromatography column box is: keeping for 5 min at 60°C, heating to
160°C at a heating rate of 25°C /min, keeping for 4 min at 160°C, heating
to 225°C at a heating rate of 2°C/min, keeping for 50 min at 225°C,
heating to 230°C at a heating rate of1C /min, and keeping for 5 min at
230°C. A detector is a flame-ionization detector (FID), a detection
temperature is 230°C, and an end-puff volume is 3.0 mL/min.
As shown in Fig. 8, the linseed oil detected by the above detection
method contains 10 fatty acids, wherein the contents of linolenic acid
(C18:3-9c, 12c, 15c), oleic acid (C18:1-9c), linoleic acid (C18:2-9c, 12c),
stearic acid (C18:0) and palmitic acid (C16:0) are higher, besides, and
also include C16:1-9c, C17:0, C18:1-11c, C20:0, C18:3-9t, 12t, 15c,
C18:3-9c, 12t, 15t, C18:3-6c, 9c, 12c, C20:1-5c, C19:2-10c, 13c,
C18:3-9t, 12c, 15c, C22:0, C20:2-11c, 14c, C20:3-8c, 11c, 14c,
C21:2-12c, 15c, and C24:0.
Comparative Example 1
A qualitative and quantitative analysis of fatty acids in milk by a gas
chromatography method, includes:
weighing 1 mL of milk in a flask, adding 5 mL of ammonia water,
mixing, hydrolyzing in a 70-80°C water bath for 20 min, shaking the
flask every 5 minutes to mix particles adhered to a wall of the flask into a
solution, cooling to room temperature after hydrolysis, adding 10 mL of
% ethanol, mixing, transferring the solution in the flask to a separating
funnel, rinsing the flask and a stopper of the flask with 50 mL of a mixed
solution of ether and petroleum ether, merging into the separating funnel,
covering, shaking for 5 min, standing for 10 min, collecting the ether
layer extract into a 250 mL flask, repeating extraction of the solution for
3 times according to the above steps, rinsing the separating funnel with
the mixed solution of ether and petroleum ether, collecting in a 250 mL
flask, concentrating with a rotary evaporator to obtain fat extract,
centrifuging at 4000 rpm for 10 min, diluting 20 pL of the supernatant in
a 1 mL volumetric flask to 1 mL, and detecting by Shimadzu GC-2010
gas chromatography. A detection condition for the gas chromatography
are: gas chromatography column: CP Sil 88 (100mx0.25mmx0.20pm), injection temperature: 270°C, an injection volume: 1 L, a split ratio:
100:1, a flow rate: 10.6 cm/s (nitrogen gas); a constant linear velocity
mode. A temperature program of a gas chromatography column box is:
keeping for 13 min at 100°C, heating to 180°C at a heating rate of 10°C
/min, keeping for 6 min at 180°C, heating to 200°C at a heating rate of
1°C/min, keeping for 20 min at 200°C, heating to 230°C at a heating rate
of 4°C /min, and keeping for 10.5 min at 230°C. A detector is a
flame-ionization detector (FID), a detection temperature is 280°C, and an
end-puff volume is 3.0 mL/min.
As shown in Fig. 9, the milk detected by the above detection method
contains 7 fatty acids, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0,
palmitic acid (C16:0). Compared with the Embodiment 2, it can be seen
that stearic acid (C18:0), oleic acid (C18:1-9c) and linoleic acid
(C18:2-9c, 12c) are not detected.
Comparative Example 2
Embodiment 3
A qualitative and quantitative analysis of fatty acids in linseed oil by
a gas chromatography method, includes:
weighing 100 mg of linseed oil in a 10 mL centrifuge tube, adding
0.1 mL of a 2 mol/L of a methanol solution of potassium hydroxide,
performing vortex mixing for 30 s, centrifuging at 4000 rpm for 10 min,
diluting 20 pL of the supernatant in a 1 mL volumetric flask to 1 mL, and detecting by Shimadzu GC-2010 gas chromatography. A detection condition for the gas chromatography are: gas chromatography column:
CP Sil 88 (100mx0.25mmx0.20pm), injection temperature: 270°C, an
injection volume: 1 L, a split ratio: 100:1, a flow rate: 10.6 cm/s
(nitrogen gas); a constant linear velocity mode. A temperature program of
a gas chromatography column box is: keeping for 13 min at 100°C,
heating to 180°C at a heating rate of10°C /min, keeping for 6 min at
180 0C, heating to 200 0C at a heating rate of1°C/min, keeping for 20 min
at 200 0C, heating to 230 0C at a heating rate of 40 C /min, and keeping for
10.5 min at 230 0 C. A detector is a flame-ionization detector (FID), a
detection temperature is 280 0C, and an end-puff volume is 3.0 mL/min.
As shown in Fig. 10, the linseed oil detected by the above detection
method contains 7 fatty acids, wherein the contents of linolenic acid
(C18:3-9c, 12c, 15c), oleic acid (C18:1-9c), linoleic acid (C18:2-9c, 12c),
stearic acid (C18:0) and palmitic acid (C16:0) are higher, besides, and
also include C18:3-6c, 9c, 12c, and C20:1-5c. Compared with the
Embodiment 2, it can be seen that C16:1-9c, C17:0, C18:1-11c, C20:0,
C18:3-9t, 12t, 15c, C18:3-9c, 12t, 15t, C19:2-10c, 13c, C18:3-9t, 12c, 15c,
C22:0, C20:2-11c, 14c, C20:3-8c, 11c, 14c, C21:2-12c, 15c, and C24:0
are not detected.
It is found from the above experiment that the detection method of
the present invention is suitable for detecting compounds containing
C3-C24 fatty acids. In addition to peanut oil, linseed oil and milk in the
above embodiments, it can also detect various edible oils enriched fatty
acids, meat foods, vegetables, milk dairy products and other substances
enriched fatty acids. It is not detailed more here due to a limited space of
the description.
The number of devices and processing scale described here are used
to simplify the description of the present invention. The application,
modification and changes of the present invention will be obvious to
those skilled in the art.
Although the embodiments of the present invention have been
disclosed above, they are not limited to the applications previously
mentioned in the specification and embodiments and can be applied in
various fields suitable for the present invention. For an ordinary skilled
person in the field, other changes may be easily achieved. Therefore,
without departing the general concept defined by the claims and their
equivalents, the present invention is not limited to particular details and
embodiments shown and described herein.
Claims (7)
1. A gas chromatography method capable of simultaneous detecting
and separating of multiple fatty acids, being characterized in that, the
method includes: detecting by gas chromatography with a CP Sil 88 gas
chromatographic column using cyanopropyl siloxane as a stationary
phase, wherein a specification of the gas chromatographic column is
-200 m x 0.25 mm x 0.20 pm, and detection conditions for the gas
chromatography include:
an injection temperature: 200-230°C; an injection volume: IpL; a
split ratio: 8-12:1; a flow rate of nitrogen gas: 10-12 cm/s; a constant
linear velocity mode;
keeping for 4-6 min at an initial temperature of 60-80°C, heating to
150-180°C at a heating rate of 20-30°C /min, keeping for 3-5 min at
150-180 0C, heating to 200-225 0C at a heating rate of 1-50 C/min, keeping
for 40-60 min at 200-225 0C, heating to 200-230C at a heating rate of
0.5-2 0C /min, keeping for 4-6 min at 200-230°C;
a detector: a flame-ionization detector (FID), a detection temperature:
200-230 0C, and an end-puff volume: 2-5 mL/min.
2. The gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids according to claim 1,
being characterized in that, the specification of the gas chromatographic
column is 100 m x 0.25 mm x 0.20pm.
3. The gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids according to claim 1,
being characterized in that, the detection conditions for gas
chromatographyinclude:
the injection temperature: 230°C; the injection volume: 1 L; the
split ratio: 10:1; the flow rate of nitrogen gas: 10.6 cm/s; the constant
linear velocity mode;
keeping for 5 min at the initial temperature of 60°C, heating to
160°C at the heating rate of 25°C /min, keeping for 4 min at 160°C,
heating to 225°C at the heating rate of 2°C/min, keeping for 50 min at
225 0C, heating to 230 0C at the heating rate of 1°C /min, and keeping for
min at 230°C;
the detector: the flame-ionization detector (FID), the detection
temperature: 230 0C, and the end-puff volume: 3 mL/min.
4. The gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids according to any one of
claims 1-3, being characterized in that, the multiple fatty acids include
C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and
C13- C24 long-chain fatty acids.
5. The gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids according to claim 4,
being characterized in that, the multiple fatty acids include C3:0, C4:0,
C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C:0, C12:0, C11:1-10c, C13:0,
C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14:1-9c, C15:0, C15:1-10t,
C15:1-10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:-10t,
C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c,
C18:1-11c, C19:0, C18:2-9t,12t, C19:1-7t, C19:1-10t, C19:1-7c,
C19:1-10c, C18:2-9c, 12c, C20:0, C20:1-11t, C18:3-6c, 9c, 12c,
C20:1-5c, C20:1-8c, C20:1-11c, C19:2-10c, 13c, C18:3-9c, 12c, 15c,
C21:0, C18:2-9c, lit, C18:2-10t, 12c, C21:1-12c, C20:2-11c, 14c, C22:0,
C22:1-13t, C20:3-8c, 11c, 14c, C22:1-13c, C21:2-12c, 15c, C20:3-11c,
14c, 17c, C23:0, C20:4-5c, 8c, 11c, 14c, C23:1-14c, C22:2-13c, 16c,
C24:0, C20:5-5c, 8c, 11c, 14c, 17c, C24:1-15c, C22:3-13c, 16c, 19c,
C22:4-7c, 10c, 13c, 16c, C22:5-4c, 7c, 10c, 13c, 16c, C22:5-7c, 10c, 13c,
16c, 19c, C22:6-4c, 7c, 10c, 13c, 16c, 19c.
6. The gas chromatography method capable of simultaneous
detecting and separating of multiple fatty acids according to claim 5,
being characterized in that, a limit of detection is 0.000084-0.001276
g/100 g, and a limit of quantitation is 0.000289-0.004263 g/100 g.
7. The gas chromatography method capable of simultaneous detecting
and separating of multiple fatty acids according to claim 5, being
characterized in that, an RSD value of intra-day accuracy is controlled
between 0.57-9.81%, and an RSD value of inter-day accuracy is
controlled between 0.47-9.87%.
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| CN111650286A (en) * | 2020-04-01 | 2020-09-11 | 上海中科新生命生物科技有限公司 | Method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry |
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| CN100509733C (en) * | 2006-08-08 | 2009-07-08 | 河南省农科院农副产品加工研究所 | Method of extracting veralkanol in peanut root using ultrasonic wave |
| CN101074188B (en) * | 2007-04-17 | 2010-07-07 | 河南省农科院农副产品加工研究所 | Method for enriching and purifying veralkcohol from peanut root by macporous adsorptive resin |
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| CN102875337A (en) * | 2011-07-13 | 2013-01-16 | 杨雨琴 | Preparation method of resveratrol |
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| CN105669386B (en) * | 2016-03-17 | 2018-08-07 | 河南省农业科学院 | A method of the separation concentration resveratrol from Roots of Peanut extracting solution |
| CN106008173A (en) * | 2016-05-22 | 2016-10-12 | 深圳市先康达生物科技有限公司 | Method for extracting anticancer resveratrol from peanut roots |
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