AU2019100394A4 - Method for detecting olefin components in orange juice through online-solid-phase micro-extraction and comprehensive two-dimensional gas chromatography/quadrupole mass spectrometer - Google Patents

Abstract

The present invention discloses a method for identifying and analyzing olefin components in orange juice through online-solid-phase micro-extraction (online-SPME) and comprehensive two-dimensional gas chromatography/quadrupole mass spectrometer (comprehensive 2DGC/qMS). The method comprises the steps: enriching olefin components in an orange juice sample through online-SPME; detecting through comprehensive 2DGC/qMS; conducting qualitative analysis on a determinand through a combination of a retention time index (LRI) and NIST spectral library; and accurately quantitatively analyzing chromatographic overlapping peaks by a nodal convolution technique for the components which cannot be separated by the comprehensive 2DGC. The comprehensive 2DGC adopts two chromatographic columns with different polarities for conducting orthogonal separation on the olefin components, and qMS is conducted under the effect of a modem for completing detection. Qualitative analysis is conducted through the combination of the LRI and the NIST spectral library, and the chromatographic overlapping peaks are accurately quantitatively analyzed by the nodal convolution technique. The method of the present invention has the characteristics of high throughput, high sensitivity and high accuracy, and solves the problems of difficult analysis and separation and difficult qualitative analysis for the olefin components in the orange juice.

Description

MICRO-EXTRACTION AND COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY/QUADRUPOLE MASS SPECTROMETER

TECHNICAL FIELD [0001] The present invention belongs to the technical field of food safety, and relates to a method for detecting olefin components in orange juice through online-solid-phase micro-extraction (online-SPME) and comprehensive two-dimensional gas chromatography/quadrupole mass spectrometer (comprehensive 2DGC/qMS).

BACKGROUD OF THE PRESENT INVENTION [0002] Orange juice has rich nutrition and fragrant and pleasant taste, and is a globally popular beverage variety which is largely consumed. The consumption of orange juice, as a main orange juice beverage, ranks the top in the world. In recent years, the orange juice and its beverage industry has developed greatly with the improvement of human recognition. However, its quality adulteration, cheating, harmful substances that exceed the standard, and other phenomena still occur occasionally due to the lack of a corresponding quality control system.

[0003] At present, the monitoring of the national standards for the safety of the orange juice mainly includes basic physical and chemical indicators, such as GB/T 21731-2008 Orange Juice and Orange Juice Beverages which specifies soluble solids, sucrose, glucose, fructose, glucose/fructose and orange juice content ratio, and penicillin. Thus, the main focus is on the basic physical and chemical indicators, pesticide residues and microbial indicators. For example, “Fanta” orange and lemon juice beverages were found to contain excessive pesticides in 2009; General Administration of Quality Supervision randomly inspected 160 kinds of fruit and vegetable juice products in August of 2011, and about 2% of orange juice beverages was not qualified due to excessive bacterial colonies, moulds and yeasts; and at the

2019100394 11 Apr 2019 beginning of 2012, Coca-Cola's Brazilian orange juice contained a small amount of carbendazim pesticide, etc. However, there is no quality standard or testing standard system for the quality of the orange juice to deal with the adulteration and blending phenomena in the orange juice industry. In 2011, the plasticizer incident of Taiwan beverage also reflected the lack of Chinese quality monitoring system for the orange juice. Therefore, the study of aromatic components in the orange juice is the basis for establishing an orange juice quality standard, and is of great significance to monitor the quality of the orange juice. Olefin components in the orange juice are important parts of the aromatic components in the orange juice, and have important to the aroma of the orange juice.

[0004] The comprehensive 2DGC/qMS is a technique which achieves two-dimensional separation of olefin substances by combining two independent chromatographic columns having different separation mechanisms in series. A modulator is arranged between the two chromatographic columns, and the modulator plays the role of capturing and transmission. In GC X GC, the separation mechanisms of two columns are independent of each other. Each fraction separated by the first chromatographic column firstly enters the modulator and is focused, and then pulsed into the second chromatographic column for further separation and analysis. Each fraction needs to be eluted into the second chromatographic column at the same time to avoid co-elution with other fractions, which may affect the separation efficiency. [0005] The existing method for detecting olefin substances in the orange juice adopts the traditional one-dimensional gas chromatography-mass spectrometry. The separation capability of the one-dimensional gas chromatography is limited so that all components cannot be separated completely. A single analysis may only analyze dozens of compounds, but the comprehensive 2DGC/qMS can detect hundreds of compounds, with a peak capacity which is hundreds to thousands of times of the one-dimensional liquid chromatography-mass spectrometry. Two-dimensional orthogonal separation can well separate the components which cannot be separated in the one-dimensional gas chromatography, thereby greatly increasing separation efficiency.

SUMMARY OF PRESENT INVENTION [0006] In view of the problems in the prior art, the purpose of the present invention to design and provide a method for detecting olefin components in orange juice

2019100394 11 Apr 2019 through online-solid-phase micro-extraction (online-SPME) and comprehensive two-dimensional gas chromatography/quadrupole mass spectrometer (comprehensive 2DGC/qMS) having high throughput, high sensitivity and good separation effect. [0007] To solve the above technical problems, the present invention adopts the following technical solution: a method for detecting olefin components in orange juice through online-SPME and comprehensive 2DGC/qMS comprises the following steps:

[0008] 1) extracting and enriching olefin components in orange juice by online-SPME;

[0009] 2) conducting orthogonal separation by comprehensive 2DGC, and detecting by qMS;

[0010] 3) conducting qualitative analysis on the olefin components through a combination of a linear retention index (LRI) and NIST spectral library; and [0011] 4) judging the purity of a chromatographic peak by a nodal convolution technique, and accurately quantitatively analyzing chromatographic overlapping peaks by different selection ions.

[0012] Specifically, the method comprises the following steps:

[0013] 1) extracting and enriching olefin components in orange juice by online-SPME [0014] weighing 5g of freshly squeezed orange juice into 10 mL of headspace sample bottle, adding 2g of NaCl, and sealing with a lid; and absorbing and desorbing samples by automatic SPME and an automatic sampling mode;

[0015] conditions of online-SPME are: an extraction head is 85 pm Polyacrylate automatic extraction head; an incubation temperature of the samples is 45°C; incubation equilibrium is conducted for 10 min; the extraction head is inserted into the sample bottle at a depth of 12 mm; and extraction time is 20 min;

[0016] 2) detecting by comprehensive 2DGC/qMS [0017] detecting olefin components by comprehensive 2DGC/qMS: extracting and enriching the olefin components by the automatic SPME; feeding determinands absorbed to the extraction head into the comprehensive 2DGC/qMS at a sample inlet through thermal desorption; separating the determinands through one-dimensional chromatographic columns; heating and cooling the determinands through a modem and then feeding into two-dimensional gas chromatographic columns for separation;

2019100394 11 Apr 2019 and conducting comprehensive scanning and detecting by qMS;

[0018] 3) conducting qualitative analysis on the olefin components [0019] a) conducting data analysis through GC image version 2.3;

[0020] b) analyzing standard n-alkanes of C8-C20 in a gas sampling mode through comprehensive 2DGC/qMS to obtain the retention time of n-alkanes of C8-C20 in one and two dimensions; and artificially defining a LRI of n-alkanes of C8-C20, for example, defining the LRI of n-alkanes with an atomic number of 8 as 800, and so on (number of carbon atoms * 100);

[0021] c) calculating the LRI of a target compound according to the retention time of the olefin components;

[0022] d) comparing a mass spectrogram of the sample with the NIST spectral library when the sample ionizes at a voltage of 0.9kv of El ionization source; and automatically identifying the substance when the similarity is greater than a set value; and [0023] e) conducting qualitative analysis on the olefin components in combination with the LRI and NIST spectral library search results;

[0024] 4) quantitatively analyzing the olefin components [0025] a) directly recording a peak area for the olefin components with good separation degree; and [0026] b) determining the peak purity for the chromatographic overlapping peaks by the nodal convolution technique; and quantitatively analyzing the overlapping peaks by selection ions when judging that the peak is formed by overlapping of multiple components.

[0027] Sample pretreatment is conducted through online-SPME in the step 1). [0028] Detection is conducted through comprehensive 2DGC/qMS in the step 2). [0029] Qualitative analysis is conducted through a combination of the LRI and NIST spectral library in the step 3).

[0030] The chromatographic overlapping peaks are accurately quantitatively analyzed by the nodal convolution technique in the step 4).

[0031] Conditions of the comprehensive 2DGC in the step 4) are as follows:

[0032] the one-dimensional chromatographic columns are nonpolar columns DB-1 15m X 0.25mm X 0.1 qm;

[0033] two-dimensional chromatographic columns are polar columns BPX-50 15m

2019100394 11 Apr 2019

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[0034] carrier gas: He, at a flow rate of 1.0 ml/mL;

[0035] sample injection conditions: analysis temperature of a sample inlet is 250 °C; analysis time is 5 min; and temperature of the sample inlet is 250 °C;

[0036] heating procedure of a column oven: an initial temperature is 35 °C for 1 min; then the temperature is raised to 270 °C at a heating rate of 3 °C /min for 5 min;

[0037] setting of the modem: a modulation period is 6s; a cold jet flow is 10 mL/min; hot jet temperature is 325 °C; and hot jet duration is 350ms; and [0038] mass spectrometry conditions: a mass spectrometry detector is a qMS; the temperature of an ion source is 230 °C; interface temperature is 280 °C; solvent delay is 3 min; and a full scanning mode is adopted, with a scanning range of 30 m/z to 300 m/z and a scanning frequency of 20000 Hz.

[0039] The present invention has the advantages and positive effects as follows: [0040] (1) One-dimensional gas chromatographic columns can only have one polarity such as nonpolarity or polarity, while nonpolar and polar columns can be used as two-dimensional gas chromatographic columns. When multiple determinands pass through the one-dimensional non-polar columns, the determinands are first separated according to molecular size, and then separated again according to polarity when passing through the two-dimensional polar chromatographic columns. Therefore, GC^XGC separation effect can be achieved so as to efficiently separate a complex matrix. Therefore, the method of the present invention is suitable for component analysis, and has absolute advantages for analyzing the olefin components in the orange juice.

[0041] (2) The qualitative analysis of an unknown substance is generally realized by means of a standard substance, and with the improvement of analysis capability of the instrument, can also be realized by means of high resolution mass spectrum. However, for component analysis, there are usually hundreds of components to be analyzed. The qualitative analysis mode of the standard substance and high resolution mass spectrum are time-consuming and expensive. Therefore, the present invention applies the LRI to the qualitative analysis of comprehensive 2DGC data: when the chromatographic columns and chromatographic conditions are the same,

2019100394 11 Apr 2019 the LRI of the substance is correlated with the number of C atoms. The present invention conducts the qualitative analysis through a combination of the LRI and

NIST spectral library.

[0042] (3) Due to the wide variety of separation substances, comprehensive 2DGC needs to be matched with mass spectrometry with high scanning speed. The qMS used in the present invention abandons the disadvantage of low scanning rate of the traditional qMS, has a scanning rate of 30000 Hz and can meet the need of comprehensive 2DGC for the scanning rate.

DESCRIPTION OF THE DRAWINGS [0043] FIG 1 is a diagram of comprehensive 2DGC/qMS of an olefin substance in an orange juice sample; and [0044] FIG 2 is a diagram of comprehensive 2DGC/qMS of n-alkane.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0045] The present invention is further described below in detail in combination with the drawings and specific embodiments.

[0046] 1. Instrument and reagent [0047] Instrument: Shimadzu Q2010 Ultra GC/GC/MS, equipped with AOC-5000 Plus automatic sampler [0048] acetonitrile, sodium chloride, analytically pure analytical reagent. Standard n-alkanes of C8-C20 (Sigma-Aldrich, ImL) [0049] 2. Sample treatment [0050] Weighing 5g of freshly squeezed orange juice into 10 mL of headspace sample bottle, adding 2g of NaCl, and sealing with a lid; and enriching and desorbing samples by automatic SPME and an automatic sampling mode. Conditions of online-SPME are: an extraction head is 85 pm Polyacrylate automatic extraction head; an incubation temperature of the samples is 45°C; incubation equilibrium is conducted for 10 min; the extraction head is inserted into the sample bottle at a depth of 12 mm; and extraction time is 20 min.

[0051] 3 Instrument conditions [0052] the one-dimensional chromatographic columns are nonpolar columns DB-1 15m X 0.25mm X 0.1 pm;

[0053] two-dimensional chromatographic columns are polar columns BPX-50 15m

2019100394 11 Apr 2019

XO.lmmXO.l μηι;

[0054] carrier gas: He, at a flow rate of 1.0 ml/mL;

[0055] sample injection conditions: analysis temperature of a sample inlet is 250 °C;

analysis time is 5 min; and temperature of the sample inlet is 250 °C;

[0056] heating procedure of a column oven: an initial temperature is 35 °C for 1 min; then the temperature is raised to 270 °C at a heating rate of 3 °C /min for 5 min;

[0057] setting of the modem: a modulation period is 6s; a cold jet flow is 10 mL/min; hot jet temperature is 325 °C; and hot jet duration is 350ms; and [0058] mass spectrometry conditions: a mass spectrometry detector is a qMS; the temperature of an ion source is 230 °C; interface temperature is 280 °C; solvent delay is 3 min; and a full scanning mode is adopted, with a scanning range of 30 m/z to 300 m/z and a scanning frequency of 20000 Hz.

[0059] Mass spectrometry conditions: ion source: El ionization source; temperature of 250 °C; transmission line temperature of 280°C; mass scanning range of 50-550amu; collection rate: 100 spectrograms per second; detector voltage of 1750V; ionization voltage of -70V. Data processing: Pegasus, spectral library search: NIST/PEST He [0060] 4 Result analysis [0061] (1) The retention times of the n-alkanes of C8-C20 under the above instrument analysis conditions are obtained, as shown in FIG. 2. In the figure, points are obtained two-dimensional chromatograms of the n-alkanes, which are C'sH C9H20, C10H22, C11H24, C12H26, C13H28, C14H30, C15H32, C16H34, C17H36, C18H38, C19H40 and C20H42 from left to right.

[0062] (2) The olefin components are absorbed through online-SPME; comprehensive 2DGC/qMS is conducted; and the olefin components in the freshly squeezed orange juice are detected. The LRIs of all detected olefin components are calculated by the retention index calculation method of GC image Version 2.3. The number of C atoms of the olefin component is preliminarily estimated based on the LRIs of the compounds according to the LRI values (as shown in Table 1).

[0063] (3) The NIST spectral library is used to perform spectral library search for olefin components which will be subjected to qualitative analysis; and fuzzy

2019100394 11 Apr 2019 qualitative analysis is performed on the detected components to obtain multiple possible structural formulas; and then final qualitative analysis is performed in combination with the number of C atoms.

[0064] (4) The peak purity is determined by the nodal convolution technique with respect to the components which cannot be separated by the comprehensive 2DGC; and the overlapping olefin components are accurately quantitatively analyzed by the selection ions.

[0065] By taking a target 1 in FIG. 1 as an example, the LRI calculated by one-dimensional retention time is 1588, so the number of C atoms of the compound is 15. Then, the top five choices after NIST spectral library search are a-Patchoulene (C15H24), Tricyclo[3.1.0.0(2,4)]hexane (Ci₂H₂₀),

2-[ 1 -(Adamantan-1 -ylamino)-2,2,2-trifluoro-ethylidene]-malononitrile (C15H16F3N3), 1,4-Methanophthalazine (CnHisN₂) and 1,3-Cyclopentadiene (CnHis). The compound is finally determined to be α-Patchoulene in combination with the number of C atoms.

Table 1 LRI of Olefin Components in Orange Juice

NO	Name	LRI	Molecular Formula	CAS
1	D-sylvestrene	1039	C10H16	1461- 27-4
2	β-Pinene	985	C10H16	127-9 1-3
3	β-Gurjunene	1502	C15H24	17334 -55-3
4	Terpinolene	1082	C10H16	586-6 2-9
5	3-Carene	1054	C10H16	13466 -78-9
6	a-Pinene	935	C10H16	80-56 -8
7	(-)-a-Panasinsen	1529	C15H24	56633 -28-4
8	1 H-Cyclopenta[ 1,3 ] cyclopropa [l,2]benzene	1385	C15H24	17699 -14-8
9	Cyclohexene, 1,5,5-trimethyl-3 -methylene-	1014	C10H16	16609 -28-2
10	Benzene, 1 -methyl-4-( 1 -methylethenyl)-	1077	C10H12	1195- 32-0
11	Perillen	1106	C10H14O	539-5 2-6

2019100394 11 Apr 2019

12	.psi.-Limonene	1000	C10H16	499-9 7-8
13	2,6-Dimethyl-1,3,5,7-octatetr a ene, E,E-	998	C10H14	460-0 1-5
14	δ-Cadinene	1524	C15H24	483-7 6-1
15	Caryophyllene	1432	C15H24	87-44 -5
16	β-Phellandrene	970	C10H16	555-1 0-2
17	β-Selinene	1459	C15H24	17066 -67-0
18	a-Gurjunene	1482	C15H24	489-4 0-7
19	β-Thujene	927	C10H16	28634 -89-1
20	β-Elemen	1396	C15H24	515-1 3-9
21	Eremophilene	1512	C15H24	10219 -75-7
22	L-calamenene	1521	C15H22	483-7 7-2
23	Germacrene D	1521	C15H24	23986 -74-5
24	β-Pinene	975	C10H16	127-9 1-3
25	a-Cubebene	1356	C15H24	17699 -14-8
26	1,3 -Cyclohexadiene, 1,3,5,5-tetramethyl-	1119	C10H16	4724- 89-4
27	Bomylene	1222	C10H16	464-1 7-5
28	1,3 -Cyclohexadiene, 1,3,5,5-tetramethyl-	1132	C10H16	4724- 89-4
29	Styrene	877	C8H8	100-4 2-5
30	β-ylangene	1439	C15H24
31	a-Gurjunene	1441	489-40-7	489-4 0-7
32	Cyclooctene, 3-(1 -methylethenyl)-	1128	C11H18	61233 -78-1
33	Cyclohexene, 2-ethenyl-1,3,3-trimethyl-	1095	C11H18	5293- 90-3
34	(E)^-Famesene	1448	C15H24	18794 -84-8
35	Limonene oxide, trans-	1125	C10H16O	4959- 35-7
36	cis-2,6-Dimethyl-2,6-octadiene	998	C10H18	2492- 22-0
37	l,E-4,Z-8-Dodecatriene	1325	C12H20	83489 -22-9
38	Limonene oxide, cis-	1121	C10H16O	13837 -75-7
39	a-Calacorene	1540	C15H20	21391 -99-1

2019100394 11 Apr 2019

40	β-Gurjunene	1475	C15H24	17334 -55-3
41	psi.-Limonene	1049	C10H16	499-9 7-8
42	2,6-Dimethyl-1,3,5,7-octatetr a ene, E,E-	1119	C10H14	460-0 1-5
43	Limonene glycol	1313	C10H18O2	1946- 00-5
44	1,4-Cyclohexadiene, 1-methyl-	759	C7H10	4313- 57-9
45	Globulol	1663	C15H26O	51371 -47-2
46	Cyclooctene, 3-(1 -methylethenyl)-	1125	C11H18	61233 -78-1
47	β-Myrcene	910	C10H16	123-3 5-3
48	.(+)-Sativen	1409	C15H24	3650- 28-0
49	trans-α-Β ergamotene	1363	C15H24	13474 -59-4
50	Naphthalene, 1,6-dimethyl-4-( 1 -methylethyl)	1668	C15H18	483-7 8-3
51	Bicyclo[7.2.0]undec-4-ene, 4,11,1 l-trimethyl-8-methylene	1416	C15H24	13877 -93-5
52	4-Acetyl-1 -methylcyclohexene	1108	C9H14O	6090- 09-1
53	1H-Indene, 1-methylene-	1170	C10H8	2471- 84-3
54	Elixene	1344	C15H24	3242- 08-8
55	a-Methylstyrene	970	C9H10	98-83 -9
56	2,4-Dimethyl-1 -heptene	843	C9H18	19549 -87-2
57	1,2,4,4-Tetramethylcyclopente ne	838	C9H16	65378 -76-9

2019100394 11 Apr 2019

The claims defining the invention are as follows:

Claims (7)

1. 2019100394 11 Apr 2019

   The claims defining the invention are as follows:

   1. A method for detecting olefin components in orange juice through online-solid-phase micro-extraction (online-SPME) and comprehensive two-dimensional gas chromatography/quadrupole mass spectrometer (comprehensive 2DGC/qMS), comprising the following steps:

   1) conducting orthogonal separation on samples by comprehensive 2DGC, and detecting by qMS;
2. 2) conducting qualitative analysis on the olefin components through a combination of a retention time index (LRI) and NIST spectral library; and
3. 3) judging the purity of a chromatographic peak by a nodal convolution technique, and accurately quantitatively analyzing chromatographic overlapping peaks by different selection ions.

   2. The method for detecting olefin components in orange juice through online-SPME and comprehensive 2DGC/qMS according to claim 1, wherein the method comprises the following steps:

   1) extracting and enriching olefin components in orange juice by online-SPME;

   weighing less than 5g of freshly squeezed orange juice into 10 mL of headspace sample bottle, adding excessive amount (about 2g) of NaCl, and sealing with a lid; and absorbing and desorbing samples by automatic SPME and an automatic sampling mode;

   2) detecting by comprehensive 2DGC/qMS detecting olefin components by comprehensive 2DGC/qMS: extracting and enriching the olefin components by the automatic SPME; feeding determinands absorbed to the extraction head into the comprehensive 2DGC/qMS at a sample inlet through thermal desorption; separating the determinands through one-dimensional chromatographic columns; then feeding the determinands into two-dimensional gas chromatographic columns after processed by a modem for separation; and conducting comprehensive scanning and detecting on the olefin components by qMS;

   3) conducting qualitative analysis on the olefin components through a combination of the LRI and NIST spectral library search results

   2019100394 11 Apr 2019

   a) conducting data analysis through GC image version 2.3;

   b) analyzing standard n-alkanes of C8-C20 in a gas sampling mode through comprehensive 2DGC/qMS to obtain the retention time of n-alkanes of C8-C20 in one and two dimensions; and artificially defining a LRI of n-alkanes of C8-C20, for example, defining the LRI of n-alkanes with an atomic number of 8 as 800, and so on (number of carbon atoms ^x 100);

   c) calculating the LRI of a target compound according to the retention time of the olefin components;

   d) comparing a mass spectrogram of the sample with the NIST spectral library when the sample ionizes at an El ionization source; and automatically identifying the substance when the similarity is greater than a set value; and

   e) conducting qualitative analysis on the olefin components in combination with the LRI and NIST spectral library search results;
4. 4) quantitatively analyzing the olefin components

   a) directly recording a peak area for the olefin components with good separation degree; and

   b) determining the peak purity for the chromatographic overlapping peaks by the nodal convolution technique; and quantitatively analyzing the overlapping peaks by selection ions when judging that the peak is formed by overlapping of multiple components.

   3. The method for detecting olefin components in orange juice through online-SPME and comprehensive 2DGC/qMS according to claim 2, wherein conditions of the comprehensive 2DGC in the step 2) are as follows:

   1) the one-dimensional chromatographic columns are nonpolar columns DB-1 15m^x0.25mm^x0.1 pm;

   2) two-dimensional chromatographic columns are polar columns BPX-50 15m^x0.1mm^x0.1 pm;

   3) carrier gas: He, at a flow rate of 1.0 ml/mL;

   4) sample injection conditions: analysis temperature of a sample inlet is 250 °C;

   analysis time is 5 min; and temperature of the sample inlet is 250 °C;
5. 5) heating procedure of a column oven: an initial temperature is 35 °C to 40 °C

   2019100394 11 Apr 2019 for 1 to 2 min; then the temperature is raised to 270 °C to 300 °C at a heating rate of 3 to 5 °C /min for 5 to 8 min;
6. 6) setting of the modem: a modulation period is 6s; a cold jet flow is 10 mL/min; hot jet temperature is 325 °C; and hot jet duration is 350ms; and
7. 7) mass spectrometry conditions: a mass spectrometry detector is a qMS; the temperature of an ion source is 230 °C; interface temperature is 280 °C; solvent delay is 3 to 4 min; and a full scanning mode is adopted, with a scanning range of 30 m/z to 300 m/z and a scanning frequency of 20000 Hz.