AU2020102961A4

AU2020102961A4 - A quantitative method for determining fatty acid methyl ester in soils and its application

Info

Publication number: AU2020102961A4
Application number: AU2020102961A
Authority: AU
Inventors: Weiwei Lu; Quanzhi Zha; Yirui Zhang
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2020-12-24
Anticipated expiration: 2028-10-22

Abstract

The invention provides a quantitative method for determining fatty acid methyl esters (FAMEs) in soils as well as its application. First, the external standard of 37 kinds of FAMEs is used to determine the operating parameters of gas chromatography-mass spectrometer (GC-MS). Second, the mixed solution of the internal standard of nonadecanoic acid methyl ester (19:0 FAME) and the external standard of 37 kinds of FAMEs is used to determine the correction factor of each FAME in the external standard. Third, the phospholipid fatty acid (PLFA) is extracted from the soil sample and methylated to FAMEs. Fourth, qualitative analysis of FAMEs with 9-19 carbon (C) atoms in the sample is performed by referring to the retention time of the external standard and the National Institute of Standards and Technology (NIST) library search. Furthermore, the correction factor of the FAMEs with 9-19 C atoms in the sample is determined by referring to the obtained correction factors of the external standard and the chemical structure of the FAMEs to be tested. Finally, the content of soil FAMEs with 9-19 C atoms is calculated based on the obtained correction factors. Therefore, this invention provides a new method to quantify FAMEs in soils, so that the soil microbial community structure can be estimated more accurately. -1/3 4) 0 ... - Figure0

Description

-1/3

4)

0

... -

Figure0

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

A quantitative method for determining fatty acid methyl ester in soils and its

application

The invention is described in the following statement:-

A quantitative method for determining fatty acid methyl ester in

soils and its application

TECHNICAL FIELD

The invention relates to the field of chemistry, in particular to compound detection

technology, especially to a GC-MS detection technology for compounds. Specifically, it is

a quantitative method for determining FAMEs in soils and its application.

BACKGROUND

Phospholipid fatty acids (PLFAs) are one of the membrane components of microbial

cells. Given that different microorganisms have different types of PLFAs, the application

of PLFAs to characterize the structure of soil microbial community has been widely used.

In order to improve the stability of the tested compound, and to lower the boiling point of

the tested compound and thus to make it easier to gasify, it is common tofirst derivatize

PLFAs into the corresponding methyl ester, namely FAMEs, which is then used for test.

The instruments used for determining FAMEs include GC, GC-MS, and liquid

chromatography-mass spectrometer (LC-MS). The Sherlock Microbial Identification

System (MIS), developed by the MIDI Corporation, USA is a software for the

identification and analysis of the types and contents of specific short-chain fatty acids (C9

C20) in microorganisms using GC for analysis (such as Agilent Company's GC-6850, 7820,

6890 and 7890). Although rapid detection of large batches of samples is possible by this

software, there are still drawbacks, such as a lack of FAME to be detected in the database

and misidentification of GC peaks. Furthermore, the price of MIDI software is relatively

high. The LC-MS can analyze the polar phosphate head of FAMEs, as well as the non- polar fatty acid side chain of FAMEs. It can display all the information carried by the phospholipid molecules of the original living cell membrane. However, since there is currently no commercialized spectral library, its application in FAMEs detection is limited.

The GC-MS can detect FAMEs with high sensitivity and low detection limit, and the

commercialized NIST library can be used for qualitative analysis, therefore, it has a good

application prospect.

At present, there are generally area normalization method, internal standard method,

and external standard method to quantify FAMEs. The area normalization method

considers the content of all peaks in the sample to be tested as 100% assuming that the

correction factors of all components are the same, and calculates the percentage of each

component based on the response value on GC. This method is easy and can eliminate

errors caused by differences in instrument status and operating conditions. However, this

method cannot obtain the absolute content of the compound in the sample to be tested. The

internal standard method directly utilizes the ratio of the response value on GC (peak area

or height) between the compound to be tested and the internal standard (usually 19:0 FAME)

and the content of internal standard to determine the content of compound to be tested. But

this method did not consider the difference in the response of different FAMEs to the

instrument. The external standard method estimates the content of FAMEs in the sample

by establishing a standard curve. However, since the commercialized standards cannot

contain all the target FAMEs in soils, this method cannot accurately quantify the FAMEs

specific to the soil.

In the article "Comparison and quantitativeoptimization ofPLFAs and mild alkaline

methylation methods in soil microbial community structure analysis" (Environmental

Chemistry (Chinese journal), 2014, 33(5): 760-764), the "internal standard method" (using

both internal and external standards) and "direct calculation method" (based on the

principle that the sample content and peak area are positively proportional) are used to

quantify soil FAMEs. They found the results from these two methods were different, so

they calculated the correction factor using the ratio of these two results, and corrected the

content of FAMEs that can only be quantified using the "direct calculation method".

Therefore, FAMEs in the sample without commercialized standards are also quantified,

and the quantification method of FAMEs is improved according to their study. However,

this method still has the following shortcomings: first, although the "internal standard

method" takes into account the different responses of different FAMEs to GC-MS, the

formula used for quantification assumes that the contents of the internal standard in the

sample and the mixed solution of internal and external standards are the same, which limits

the application of the formula; second, the correction factor of the FAMEs that is absent in

the external standard is set to be the average correction factors of all the known FAMEs,

which can increase the error to some extent. Therefore, it is necessary to further optimize

the quantitative method of FAMEs in soils.

SUMMARY

The main purpose of the invention is to overcome the shortcomings in previously

established methods and to establish a quantitative method for determining FAMEs in soils,

and thus to characterize the soil microbial community structure more accurately.

One of the objectives of the invention is to provide a quantitative method for

determining FAMEs in soils, which includes the following steps:

(1) Determine the operating parameters on GC-MS: analyze the external standard of

37 kinds of FAMEs on GC-MS to determine its operating parameters;

(2) Determine the correction factor of each FAME in the external standard: prepare a

mixed solution which contains internal standard (19:0 FAME) and external standard and

analyze the mixed solution on the GC-MS using the operating parameters determined in

step (1). The correction factor of each FAME in the external standard is calculated as

follows:

MX/AX

where, is the correction factor of the FAME in the external standard; Mo is the mass

of the internal standard (pg); Ao is the response value of the internal standard on the GC;

Ax is the response value of the target FAME on the GC; M, is the mass of the target FAME

(g).

(3) Extract PLFAs from the soil and methylate them to FAMEs: collect soil samples,

extract PLFAs from the soil using the Bligh-Dyer method, and methylate the extracted

PLFAs to FAMEs.

(4) Perform qualitative analysis of FAMEs in the soil samples: Add internal standard

19:0 FAME to the soil FAMEs obtained in step (3), analyze the samples by GC-MS using

the operation parameters determined in step (1), and carry out qualitative analysis of soil

FAMEs with 9-19 C atoms. Determine the correction factors of the soil FAMEs with 9-19

C atoms.

(5) Perform quantitative analysis of FAMEs in the soil samples: calculate the concentration of FAMEs (C, nmol g-1) with 9-19 C atoms in soil using the correction factors determined in step (4) and the response value of each FAME on GC using the following equation:

Mi A 1000 C=fx-x x Ai MmoI m

wherefis the correction factor of the FAME to be tested in soil; Mi is the mass of the

internal standard (g); Ai is the response value of the internal standard on the GC; A is the

response value of the FAME on the GC; Mmoi is the molecular molar mass of the FAME to

be tested ( g mol-1); and m is the mass of soil on oven-dried basis (g).

As a preference, the determination of the GC-MS operating parameters in step (1)

includes the following process:

1) Dilute 0.08 ml of the purchased external standard to 2 ml with n-hexane, that is, 25

times dilution. The final concentrations of the diluted external standard range from 6.58 to

24.9 g ml-1 and then they are analyzed on GC-MS;

2) For GC analysis, a VF-23 ms chromatographic column is selected to determine

FAMEs. The carrier gas is helium. The injection is split-less with the injection volume of

1 , and the temperature of the injection port is 260 °C; the heating program of the

chromatographic column is as follows: the initial temperature of 60 °C for 1 min; heating

at 5 °C min-' to 170 °C for 2 min; and heating at 2 °C min-' to 200 °C for 5 min.

3) For MS analysis, full scan mode with the scanning range of 50-500 (m/z ratio) is

used. The MS is operated in electron ionization (El) mode with the electron energy of 70

eV. The temperature of ion source and transfer line is set at 180 °C and 200 °C, respectively.

As preferred, the procedure of preparing a mixed solution of internal standard (19:0

FAME) and external standard in step (2) is as follows: weigh 0.002 g of internal standard

19:0 FAME and use 10 ml of n-hexane to prepare the internal standard solution with the

final concentration of 200 g ml- 1; mix 0.2 ml of 200 g ml-' internal standard solution

with 0.2 ml of the purchased external standard solution, and dilute the mixture to 5 ml with

n-hexane to obtain a mixed solution with the internal standard concentration of 8 g ml-'

and the external standard concentration of 6.58-24.9 g ml- 1. Add 2 ml of the mixed

solution into the GC vial for GC-MS analysis.

Preferably, the Bligh-Dyer method used to extract PFLAs in soils described in step (3)

includes the following procedures:

a. Extract soil lipids: weigh fresh soil of 2.00 g on oven-dried basis. Extract the fresh

soil with 15 ml Bligh-Dyer solution and collect the extract solution, and then re-extract the

soil with 7 ml Bligh-Dyer solution. Combine the two extract solutions and leave overnight

in order to make the soil lipid thoroughly dissolved in the chloroform. Collect the

underneath chloroform layer the next day and dry it under N2 blowing in a water bath of

37 °C;

b. Separate the PLFAs: dissolve the soil lipids obtained in step (a) with chloroform,

then add them to a silica gel column. Elute the silica gel column using 5 ml chloroform, 5

ml acetone and 5 ml methanol sequentially. Collect the effluent eluted by the methanol and

dry it under N2 blowing in a water bath of 37 C to obtain PLFAs;

c. Methyl esterification reaction: add 1 ml 1:1 (v/v) toluene-methanol solvent to

dissolve the PLFAs obtained in step b; then add 1 ml 0.2 mol - methanol-KOH solution, and react for 30 min in a water bath of 37 °C; finally add 1 ml 1 mol 1-' acetic acid to stop the reaction; then add 2 ml chloroform, 2 ml water and leave overnight to form layers; and then collect the organic solution layer, and dry it under N2 blowing in a water bath of 37

°C to obtain the FAMEs to be tested.

The ratio of chloroform, methanol and phosphate buffer in the Bligh-Dyer solution in

step (a) is 1:2:0.8 (v/v/v).

Preferably, the concentration of the internal standard solution added to the FAMEs to

be tested in step (4) is 3.2 g ml-1 , and its volume is 0.5 ml.

Preferably, the qualitative analysis described in step (4) refers to the qualitative

analysis of FAMEs with 9-19 C atoms to be tested by reference of the retention time of the

external standard, literature and NIST library search.

Preferably, the method to determine the correction factor of the FAMEs with 9-19 C

atoms in the sample to be tested described in step (4) is as follows: if the FAME present in

the external standard, its correction factor is estimated as described in step (2); if the FAME

absent in the external standard, its correction factor is the same with that of the FAME

present in the external standard with the same number and position of C atom, double bond

and branch chain.

Another object of the invention is to provide the application of the quantitative method

for determining FAMEs in soils as described above.

Compared with the existing technology, the invention has the following beneficial

effects:

The invention utilizes the internal and the external standard at the same time, considers

the different responses of different FAMEs to the instrument, and obtains the correction

factors of the target FAMEs in soil samples which is absent in the external standard, and

thus quantify the FAMEs more accurately. The application of the invention can optimize

the characterization of soil microbial community structure by using PLFAs technology,

moreover, it also can be applied to determine the FAMEs of various C chain lengths in

other samples.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 The chromatogram of the external standard of 37 kinds of FAMEs used in

the invention;

Figure 2 The technical flow chart of the invention;

Figure 3 The proportions of soil microbial groups obtained by quantifying FAMEs

using the invention.

DESCRIPTION OF THE INVENTION

In order to understand the invention, it is further described in combination with the

embodiment.

The materials and experimental methods involved in the invention are described as

follows:

Apparatus: gas chromatography-mass spectrometer (GC-MS, Claus 680-SQ8T, PE

Co., USA).

Reagents: external standard of 37 kinds of FAMEs is purchased from Sigma-Aldrich

Company; internal standard of 19:0 FAME is purchased from Accustandard Company; n

hexane and methanol are of chromatographic grade and are purchased from Tedia

Company; Toluene is of chromatographic grade and are purchased from Fisher Scientific

Company; Acetone and chloroform are both of HPLC grade, and are purchased from

Nanjing Chemical Reagent Ltd.; 3 ml silica gel solid phase extraction (SPE) column is

purchased from Waters Company.

Materials: extraction device, PTFE centrifuge tube, brown screw bottles of different

sizes (40 ml, 15 ml, 4 ml and 1.5 ml), long burette, measuring cylinder, beaker, etc.

Experiment 1: Determination of operating parameters of GC-MS

24.9 g ml-1 and then they are analyzed on GC-MS;

2) For GC analysis, a VF-23 ms chromatographic column is selected to determine

3) For MS analysis, full scan mode with the scanning range of 50-500 (mlz ratio) is

used. The MS is operated in electron ionization (El) mode with the electron energy of 70 eV. The temperature of ion source and transfer line is set at 180 °C and 200 °C, respectively.

The chromatography of the external standard of 37 kinds of FAMEs is shown in Fig.

1.

Experiment 2: Determine the correction factor of each FAMEs in the external standard

Prepare a mixed solution which contains internal standard (19:0 FAME) and external

standard and analyze the mixed solution on the GC-MS using the operating parameters

determined in step (1). The correction factor of each FAME in the external standard is

calculated as follows:

wherefr is the correction factor of the FAME in the external standard; Mo is the mass

of the internal standard (kg); Ao is the response value of the internal standard on the GC;

(pg).

The procedure of preparing a mixed solution of internal standard (19:0 FAME) and

external standard is as follows: weigh 0.002 g of internal standard 19:0 FAME and use 10

ml of n-hexane to prepare the internal standard solution with the final concentration of 200

g ml- 1; mix 0.2 ml of 200 g ml-' internal standard solution with 0.2 ml of the purchased

external standard solution, and dilute the mixture to 5 ml with n-hexane to obtain a mixed

solution with the internal standard concentration of 8 g ml-' and the external standard

concentration of 6.58-24.9 g ml-1. Add 2 ml of the mixed solution into the GC vial for

GC-MS analysis

The correction factors of FAMEs in the external standard are shown in Table 1.

Table 1

FAME CAS NO. Corresponding PLFAs Retention Correction factor

time (min)

Methyl butyrate 623.42.7 4:0 -

Methyl hexanoate 106.70.7 6:0 4.14 2.22

Methyl octanoate 111.11.5 8:0 7.20 1.14

Methyl decanoate 110.42.9 10:0 10.79 0.96

Methyl undecanoate 1731.86.8 11:0 12.58 0.91

Methyl laurate 111.82.0 12:0 14.32 0.84

Methyl tridecanoate 1731.88.0 13:0 15.97 0.83

Methyl tetradecanoate 124.10.7 14:0 17.56 0.77

Myristoleic acid methyl ester 56219.06.8 14:1o5c 18.36 0.84

Methyl pentadecanoate 7132.64.1 15:0 19.06 0.78

Cis-10-pentadecenoic acid methyl ester 90176.52.6 i15:1o5t 19.85 0.81

Methyl palmitate 112.39.0 16:0 20.50 0.72

Methyl palmitoleate 1120.25.8 16:1o7c 21.08 0.79

Methyl heptadecanoate 1731.92.6 17:0 21.86 1.04

Cis-10-heptadecenoic acid methyl ester 75190.82.8 17:1o7c 22.44 0.80

Methyl octadecanoate 112.61.8 18:0 23.17 0.76

Trans-9-elaidic acid methyl ester 1937.62.8 18:1o9t 23.49 0.83

Cis-9-oleic acid methyl ester 112.62.9 18:1o9C 23.68 0.77

Linolelaidic acid methyl ester 2566.97.4 18:2o6t(6,9) 24.21 0.90

Methyl linoleate 112.63.0 18:2o6c(6,9) 24.73 0.87

y -linolenic acid methyl ester 16326.32.2 18:3o6c(6,9,12) 25.50 0.96

Methyl linolenate 301.00.8 18:3o3c(3,6,9) 26.14 1.00

Methyl arachidate 1120.28.1 20:0 26.41 0.88

Methyl cis-11-eicosenoate 2390.09.2 20:1 o9c 27.11 0.99

Methyl heneicosanoate 6064.90.0 21:0 28.29 1.15

11,14-eicosadienoic acid methyl ester 2463.02.7 20:2o6c(6,9) 28.45 1.11

Cis-8,11,14-eicosatrienoic acid methyl 21061.10.9 20:3o6c 29.34 1.21

ester

Methyl cis-5,8,11,14-eicosatetraenoic 2566.89.4 20:4o6c 29.96 1.33

Cis -11,14,17-eicosatrienoic acid methyl 55682.88.7 20:3o3c 30.08 1.68

ester

Methyl docosanoate 929.77.1 22:0 30.28 1.28

Methyl erucate 1120.34.9 22:1o9c 31.10 1.53

Methyl cis-5,8,11,14,17-eicosapentaenoate 2734.47.6 20:5o3c(3,6,9,12,15) 31.71 1.31

Methyl tricosanoate 2433.97.8 23:0 32.33 1.91

Cis-13,16-docosadienoic acid methyl ester 61012.47.3 22:2o6c(6,9) 32.60 2.07

Methyl lignocerate 2442.49.1 24:0 34.45 2.82

Methyl nervonate 2733.88.2 24:1o9c 35.36 3.39

All cis-4,7,10,13,16,19-docosahexaenoate 2566.90.7 22:6o3c(3,6,9,12,15,18) 36.95 2.19

Experiment 3: PLFAs extraction from soil and methyl esterification

37 °C;

dry it under N2 blowing in a water bath of 37 C to obtain PLFAs; c. Methyl esterification reaction: add 1 ml 1:1 (v/v) toluene-methanol solvent to dissolve the PLFAs obtained in step b; then add 1 ml 0.2 mol - methanol-KOH solution, and react for 30 min in a water bath of 37 °C; finally add 1 ml 1 mol 1 acetic acid to stop the reaction; then add 2 ml chloroform, 2 ml water and leave overnight to form layers; and then collect the organic solution layer, and dry it under N2 blowing in a water bath of 37

°C to obtain the FAMEs to be tested.

step (a) is 1:2:0.8 (v/v/v).

Embodiment 1: Qualitative analysis of FAMEs in soil

The soil samples were collected from a Zelkova Serrataplantation in Xiashu County,

Zhenjiang City, Jiangsu Province, China, an old and a young poplar plantations in Dongtai

Forest Farm, Yancheng City, Jiangsu Province, China. The soil depth was 0-20 cm. The

following procedures were adopted: Obtain the FAMEs to be tested using the methods of

soil PLFAs extraction and methyl esterification described in Experiment 3. Add 0.5 ml

internal standard (19:0 FAME) solution with the concentration of 3.2 g ml-1 to the FAMEs

to be tested. Analyze the FAMEs on GC-MS using the operating parameters determined in

Experiment 1. Perform qualitative analysis of FAMEs with 9-19 C atoms in soil samples

by referring to the retention time of the external standard determined in Experiment 2,

literature and NIST library search. Determine the correction factor of FAMEs with 9-19 C

atoms in soil samples according to the correction factors of FAMEs in external standard

determined in Experiment 2. The correction factor of FAMEs in soil samples which are

absent in the external standard is to utilize the correction factor of the FAME present in the external standard with the same number and position of C atom, double bond and branch chain.

The results of qualitative analysis and correction factors of FAMEs in soil samples

are shown in Table 2. There were a total of 24 FAMEs detected in the soils, a total of 10

FAMEs were present in the external standard while a total of 14 FAMEs were absent in

the externalstandard.

Table 2

FAMEs CAS NO. Corresponding Retention time Correction

PLFAs (min) factor

Methyl 12- methyl tridecanoate 5129.58.8 i14:0 16.85 0.77

Methyl tetradecanoate 124.10.7 14:0 17.59 0.77

Methyl 13-methyl tetradecanoate NIST#: 336314 i15:0 18.38 0.78

Methyl 12-methyl tetradecanoate 5129.66.8 a15:0 18.65 0.78

Methyl pentadecanoate 7132.64.1 15:0 19.09 0.78

Methyl 14- methyl pentadecanoate 5129.60.2 i16:0 19.84 0.72

Methyl palmitate 112.39.0 16:0 20.55 0.72

Methyl palmitelaidate 10030.74.7 16:1wo7t 20.80 0.79

Methyl 10-methyl-hexadecanoate 900336.50.9 10Me17:0 20.96 0.80

Methyl palmitoleate 1120.25.8 16:1o7c 21.13 0.79

Methyl 15-methyl hexadecanoate 6929.04.0 i17:0 21.24 0.80

Methyl trans-9-hexadecenoate 822.05.9 16:1o5 21.31 0.79

Methyl 14-methyl hexadecanoate 2490.49.5 a17:0 21.50 0.80

Methyl 10-methyl heptadecanoate 2490.25.7 1OMe18:0 21.61 0.76

Methyl cis-9-heptadecenoate 900336.38.0 17:lo8c 21.75 0.80

Methyl heptadecanoate 1731.92.6 17:0 21.90 1.04

Cis-10-heptandecanoic acid methyl ester NIST#: 333621 17:1o7c 22.43 0.80

Methyl 8-(2-hexylcyclopropyl) octanoate 10152.61.1 cyl7:0o(7,8) 22.56 0.80

Methyl octadecanoate 112.61.8 18:0 23.21 0.76

Trans-9-elaidic acid methyl ester 1937.62.8 18:1o9t 23.73 0.83

Cis-9-oleic acid methyl ester 112.62.9 18:1o9c 23.86 0.77

Cis-13-octadecenoic acid methyl ester 900333.58.3 18:lo5t 24.07 0.77

Linolelaidic acid methyl ester 2566.97.4 18:2co6t(6,9) 24.23 0.90

Cis-10-nonadecenoic acid methyl ester 900333.64.4 19:1 o9c 25.47 1.00

Embodiment 2: Quantitative analysis of FAMEs in soil samples

The concentration of the FAMEs in the soil (C, nmol g-') is calculated according to

the correction factors of various FAMEs obtained in Example 1, the response values of the

internal standard and each FAME on GC using the following formula:

M. A 1000 C=fx-x x Ai Mmoi m

be tested ( g mol-'); and m is the mass of soil on oven-dried basis (g).

The content of PLFAs (nmol g-') used to characterize the microbial community

structure in different soil samples are shown in Table 3.

Table 3

Old poplar Young poplar plantation Zelkova Serrata PLFAs plantation soil soil plantation soil

i14:0 0.05 0.03 0.02

14:0 0.09 0.05 0.05

i15:0 0.94 1.35 0.41

a15:0 0.49 0.25 0.22

15:0 0.09 0.06 0.06

i16:0 0.39 0.22 0.18

16:0 2.38 1.75 1.37

16:l1wc7t 0.11 0.07 0.05

lOMel7:0 1.00 0.65 0.56

16:lwo7c 0.95 1.23 0.32

i17:0 0.32 0.36 0.20

16:1co5 0.56 0.37 0.26

a17:0 0.36 0.22 0.18

lOMel8:0 0.18 0.11 0.15

17:lwo8c 0.47 0.36 0.27

17:0 0.20 0.17 0.14

17:lwo7c 0.53 0.36 0.28

cyl7:Oco(7,8) 0.09 0.06 0.07

18:0 0.71 0.39 0.37

18:lw09t 0.88 0.65 0.36

18:lw09c 1.49 0.69 0.54

18:lcot/7t 0.15 0.12 0.05

18:2wo6t(6,9) 0.04 0.04 0.00

19:lw09c 0.92 0.63 0.41

Total 13.4 10.2 6.50

Embodiment 3: Analysis of soil microbial community structure

Soil microorganisms are divided into five groups: general bacteria, G- bacteria, G'

bacteria, fungi, and actinomycetes according to PLFAs biomarkers. The proportions of

microbial groups in the soil samples from Zelkova Serrataplantation, and old and young

poplar plantations are shown in Figure 3.

Although the present invention has been disclosed in better embodiments, it is not

intended to constrain the invention. Any technical personnel familiar with the art can make

some changes or modifications by utilizing the technical contents disclosed above without

departing from the technical scheme of the invention. Any simple modification, equivalent

change and modification to the above embodiment according to the technical essence of

the invention still belongs to the scope of the technical solution of the invention.

Claims

THE CLAIMS:

1. A quantitative method for the determining FAMEs in soils is characterized in that

it comprises the following steps:

(1) Determine the operating parameters on GC-MS: analyze the external standard of

37 kinds of FAMEs on GC-MS to determine its operating parameters;

(2) Determine the correction factor of each FAME in the external standard: prepare a

mixed solution which contains internal standard (19:0 FAME) and external standard and

analyze the mixed solution on the GC-MS using the operating parameters determined in

step (1). The correction factor of each FAME in the external standard is calculated as

follows:

where, is the correction factor of the FAME in the external standard; Mo is the mass

of the internal standard (pg); Ao is the response value of the internal standard on the GC;

Ax is the response value of the target FAME on the GC; M, is the mass of the target FAME

(pg).

(3) Extract PLFAs from the soil and methylate them to FAMEs: collect soil samples,

extract PLFAs from the soil using the Bligh-Dyer method, and methylate the extracted

PLFAs to FAMEs.

(4) Perform qualitative analysis of FAMEs in the soil samples: Add internal standard

19:0 FAME to the soil FAMEs obtained in step (3), analyze the samples by GC-MS using

the operation parameters determined in step (1), and carry out qualitative analysis of soil

FAMEs with 9-19 C atoms. Determine the correction factors of the soil FAMEs with 9-19

C atoms.

(5) Perform quantitative analysis of FAMEs in the soil samples: calculate the

concentration of FAMEs (C, nmol g-') with 9-19 C atoms in soil using the correction factors

determined in step (4) and the response value of each FAME on GC using the following

equation:

M. A 1000 C~fx-x x Ai MmoI m

wherefis the correction factor of the FAME to be tested in soil; Mi is the mass of the

internal standard (g); Ai is the response value of the internal standard on the GC; A is the

response value of the FAME on the GC; Mmoi is the molecular molar mass of the FAME to

be tested ( g mol-'); and m is the mass of soil on oven-dried basis (g).

2. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that: the determination of GC-MS operating parameters in step (1) includes

the following procedures:

1) Dilute 0.08 ml of the purchased external standard to 2 ml with n-hexane, that is, 25

times dilution. The final concentrations of the diluted external standard range from 6.58 to

24.9 g ml-' and then they are analyzed on GC-MS;

2) For GC analysis, a VF-23 ms chromatographic column is selected to determine

FAMEs. The carrier gas is helium. The injection is split-less with the injection volume of

1 , and the temperature of the injection port is 260 °C; the heating program of the

chromatographic column is as follows: the initial temperature of 60 °C for 1 min; heating at 5 °C min-' to 170 °C for 2 min; and heating at 2 °C min-' to 200 °C for 5 min.

3) For MS analysis, full scan mode with the scanning range of 50-500 (mlz ratio) is

used. The MS is operated in electron ionization (El) mode with the electron energy of 70

eV. The temperature of ion source and transfer line is set at 180 °C and 200 °C, respectively.

3. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that: the procedure of preparing a mixed solution of internal standard (19:0

FAME) and external standard in step (2) is as follows: weigh 0.002 g of internal standard

19:0 FAME and use 10 ml of n-hexane to prepare the internal standard solution with the

final concentration of 200 g ml-1; mix 0.2 ml of 200 g ml-1 internal standard solution

with 0.2 ml of the purchased external standard solution, and dilute the mixture to 5 ml with

n-hexane to obtain a mixed solution with the internal standard concentration of 8 g ml-1

and the external standard concentration of 6.58-24.9 g ml-1. Add 2 ml of the mixed

solution into the GC vial for GC-MS analysis.

4. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that the Bligh-Dyer method used to extract PFLAs in soils described in

step (3) includes the following procedures:

a. Extract soil lipids: weigh fresh soil of 2.00 g on oven-dried basis. Extract the fresh

soil with 15 ml Bligh-Dyer solution and collect the extract solution, and then re-extract the

soil with 7 ml Bligh-Dyer solution. Combine the two extract solutions and leave overnight

in order to make the soil lipid thoroughly dissolved in the chloroform. Collect the

underneath chloroform layer the next day and dry it under N2 blowing in a water bath of

37 °C; b. Separate the PLFAs: dissolve the soil lipids obtained in step (a) with chloroform, then add them to a silica gel column. Elute the silica gel column using 5 ml chloroform, 5 ml acetone and 5 ml methanol sequentially. Collect the effluent eluted by the methanol and dry it under N2 blowing in a water bath of 37 °C to obtain PLFAs; c. Methyl esterification reaction: add 1 ml 1:1 (v/v) toluene-methanol solvent to dissolve the PLFAs obtained in step b; then add 1 ml 0.2 mol - methanol-KOH solution, and react for 30 min in a water bath of 37 °C; finally add 1 ml 1 mol 1 acetic acid to stop the reaction; then add 2 ml chloroform, 2 ml water and leave overnight to form layers; and then collect the organic solution layer, and dry it under N2 blowing in a water bath of 37

°C to obtain the FAMEs to be tested.

5. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that: the concentration of the internal standard solution added to the

FAMEs to be tested in step (4) is 3.2 g ml-1 , and its volume is 0.5 ml.

6. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that: the qualitative analysis described in step (4) refers to the qualitative

analysis of FAMEs with 9-19 C atoms to be tested by reference of the retention time of the

external standard, literature and NIST library search.

7. The quantitative method for determining FAMEs in soils according to claim 1 is

characterized in that: the method to determine the correction factor of the FAMEs with 9

19 C atoms in the sample to be tested described in step (4) is as follows: if the FAME

present in the external standard, its correction factor is estimated as described in step (2); if the FAME absent in the external standard, its correction factor is the same with that of the FAME present in the external standard with the same number and position of C atom, double bond and branch chain.

8. The application of the quantitative method for determining FAMEs in soils

according to any claim of 1-6.

Figure 1 -1/3-

-2/3 22 Oct 2020

Determine the operating parameters of GC-MS

using the external standard of 37 kinds of FAMEs

Determine the correction factor of each FAME in 2020102961

the external standard using the mixed solution of

internal standard and external standard

Extract PLFAs from the soil and methylate them to

FAMEs, and add internal standard to soil FAMEs

Perform qualitative analysis of soil FAMEs with

9-19 C atoms by referring to the retention time of

the external standard and the NIST library search,

and determine the correction factors of the FAMEs

Calculate the concentration of soil FAMEs with 9-

19 C atoms using the correction factors

Figure 2

-3/3- 22 Oct 2020

General bacteria Gram-negative bacteria Gram-positive bacteria 0.8 Bacteria Fungi Actinomycetes

0.7

0.6 2020102961

Proportion (%)

0.5

0.4

0.3

0.2

0.1

0.0 Young poplar Old poplar Zelkova Serrata plantation soil plantation soil plantation soil

Figure 3