CN104101659B - A kind of construction method of hairtail finger-print - Google Patents
A kind of construction method of hairtail finger-print Download PDFInfo
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Abstract
The present invention relates to a kind of construction method of hairtail finger-print, hairtail is prepared into need testing solution, do all one can to detect through high performance liquid chromatograph, obtain the finger-print of hairtail; Described method comprises the determination of the preparation of need testing solution, high-performance liquid chromatogram determination, standard finger-print.The present invention utilizes HPLC chromatography to establish the finger-print of hairtail, and deemed-to-satisfy4 investigation shows that the method is reliable, repeatability is better, meets the requirement of finger-print; The present invention detects that the method in the hairtail place of production is simple to operate, detection efficiency is high, testing result good reliability, fast, degree of accuracy is high, and accuracy rate is up to more than 92.7%.
Description
Technical field
The present invention relates to a kind of simple to operate, detection efficiency is high, testing result good reliability, fast, the construction method of the high hairtail finger-print based on high performance liquid chromatography of degree of accuracy.
Background technology
Hairtail (Trichiuruslepturus), also known as dental strip, leukorrhea fish, hairtail etc., belong to net-rope Perciformes Trichiuridae animal, for lower floor's mass migration fish in warm warm nature, inhabit depth of water 60-100m shale seabed, with various fish, shrimp and cuttlefish etc. for Major Foods, have obvious Diel Vertical Migration phenomenon, be distributed widely in the temperature in the world, Tropical Ocean Area (with the Indian Ocean and Western Pacific in the majority).
Hairtail fish body is significantly flat-sided, and prolongation becomes band, and tail is elongated as whip, general body long 60 ~ 120 centimetres, body weight 200 ~ 400 grams.Long narrow and flat-sided, front end point is prominent.Two jaw teeth are flourishing and sharp-pointed.Eye is large, position is higher, smooth, the middle nick of interorbital space.Body surface smooth, squama is degenerated to epidermis silverskin, and whole body is rich glossiness silvery white, and back and dorsal fin, pectoral fin show slightly cinerous.Dorsal fin is long, and starting point upper angle after gill opening is long together along back, and anal fin is not obvious, only has fin spine to sting point and exposes.Without abdomeinal fin.
The tender body of the band flesh of fish is fertile, delicious flavour, and only have a middle large bone, without other spinelets, instant, is of high nutritive value, and is extremely a kind of marine fishes of liking of people.There is the effects such as qi-restoratives, removing toxic substances, hemostasis, can be used for the diseases such as body void, galactosis deficiency, traumatism and bleeding.Meanwhile, the caffeine extracted from hairtail epidermis silverskin, can be cooked photographic industry and medical industry raw material; The 6-thioguanine extracted is made medicine or controls acute leukemia, cancer of the stomach and lympha tumour etc., and the band flesh of fish also can produce injection of fish.
Hairtail is one of large economic fish of China four, and China's the East China Sea and the Yellow Sea, the Bohai Sea, the South Sea and the North Sea are all produced, marine products amount maximum (70%, 2006) wherein, and South Sea output is less, and Zhejiang, Along The Coast of Shandong Province are the more sea areas of output.Before nineteen ninety, East China Sea region peak annual output is 52.8 ten thousand tons (1974), and Zhejiang Province is 340,000 tons, accounts for 64% of East China Sea region, and after 1974, output starts landslide, and the whole province's output, between 20-30 ten thousand tons, is down to minimum in 1989, only has 19.5 ten thousand tons.Started from 1989; enforcement is laid eggs and is with protected location; and develop off-lying sea band fish resource; nineteen ninety-five starts again to lie prostrate season trawlnet and Movable stow net season and area closures; hairtail increase of production since being the nineties is very fast, rises to 580,000 tons, top all previous records from 240,000 tons of nineteen ninety-fives of nineteen ninety; within 2000, reach 650,000 tons, within 2010, reach 1,180,000 tons.But, the growth of band fish crop, and do not mean that the improvement of hairtail resource, the individual miniaturization of hairtail, become younger serious, winter fishing season captured population when age fish ratio from 1967 20.5%, nineteen eighty-two rises to 59.3%, the nineties is average out to 71.1% in earlier stage, the later stage nineties reaches 78.2%, reaches 82.3% at present, and visible hairtail captured population is based on the captured population when age, growth with fish crop fishes for gained by strengthening, and hairtail resource is in growth type overfishing state.
Zhoushan hairtail, geographic position of marine site uniqueness and hydrological environment residing for it, freshness and quality are compared with other areas and are come out top, and are the owners of the first batch of seafood geographical sign certification mark in the whole nation.But some illegal retailers, for seeking interests, often pretend to be Zhoushan hairtail with the hairtail in other places of production, adulterate, grievous injury consumer's interests, destroy the goodwill of Zhoushan seafood.
In in September, 1997 deliver aquatic product journal the 21st volume the 3rd interim have one section about Trichiurus Haumela From The East China Sea Population identification research report, by to sampling the observation of every morphological characters of the 29 batches of 2168 tail hairtail fish bodies in different sea areas in the East Sea, measurement application of mathematical method carries out cluster analysis between statistical and colony to Traits change, comprehensively analyze in conjunction with interrelated data.Result shows: Trichiurus Haumela From The East China Sea, no matter the colony of coastal waters or off-lying sea, mutual morphological feature exactly likes substantially, presents close relationship, thus confirms that they are same population.This method detects the hairtail place of production or the hairtail population time spent of a specified duration.
Summary of the invention
The object of the invention is to determine to solve existing hairtail place of production difficulty, do not have a kind of effectively detect hairtail place of production method fast defect and provide a kind of simple to operate, detection efficiency is high, testing result good reliability, fast, the construction method of the high hairtail finger-print based on high performance liquid chromatography of degree of accuracy.
To achieve these goals, the present invention is by the following technical solutions:
A construction method for hairtail finger-print, is prepared into need testing solution by hairtail, does all one can to detect, obtain the finger-print of hairtail through high performance liquid chromatograph;
Described method comprises the steps:
A) preparation of need testing solution: the hairtail sample getting Different sources, with Extraction solvent refluxing extraction, centrifugal, get supernatant to be added on alkali alumina chromatographic column, collect post liquid, then with water elution, merged post liquid and water elution liquid, filtered, as need testing solution;
B) high-performance liquid chromatogram determination: the need testing solution that aspiration step a) obtains measures to high performance liquid chromatograph, obtains the hairtail finger-print of Different sources;
C) determination of standard finger-print: the hairtail determining 5 Different sources totally 28 hairtail sample efficient liquid-phase chromatograph finger print atlas comparing, obtain the hairtail sample efficient liquid-phase chromatograph finger print atlas be made up of its total peak, there are 13 and 7 characteristic peaks in the common characteristic peak that this finger-print has.
In the technical program; the nutritional labeling of aquatic products and content and specific aquatic environment closely related, the identical product of kind may the place of production different, the Different sources product feature factor is analyzed; be conducive to the formation of original producton location selling point, and formulate corresponding safeguard measure further.Report in document that more is utilize high performance liquid chromatography (HPLC) technology to study TCD place of production and the true and false, traditional Chinese medicine fingerprint is the new technology of modern Chinese herbal medicine quality control and Quality Identification, for sample quality evaluation problem and place of production attaching problem provide new method and thinking, and utilize high performance liquid chromatography less to the research of aquatic products finger-print.
HPLC chromatography degree of separation is high, the chemical composition of complexity can be carried out being separated and analyzing, the different chromatographic peak of these height contains numerous information, go out the position at peak and height (or peak area) represent respectively particular chemicals and content thereof number, so whole chromatogram can characterize chemical composition contained by this sample number and the size of content.
Respectively each place of production hairtail HPLC chromatogram is imported similarity evaluation A version (2004) (Chinese Pharmacopoeia Commission), set up each place of production hairtail HPLC finger-print common pattern.According to similarity evaluation, obtain each place of production hairtail HPLC reference fingerprint collection of illustrative plates, establish 13 total peaks and 7 characteristic peaks, the comparison of peak relative retention time and relative peak area is mainly had by each place of production hairtail, find that sample room relative retention time difference is less, and relative peak area has significant difference, describe each place of production hairtail contain material have at least more than 13 kinds identical, but chemical composition content also exists difference because of marine site difference.
As preferably, in step a) hairtail sample choice body surface be rich in gloss, the cheek be scarlet, mucus is transparent, eyeball is full, cornea hairtail clearly, gets 1.5-5.0g hairtail muscle of back sample dry 12-18h at 0 to-10 DEG C, for subsequent use; Then the deionized water and stirring adding 5-6 times of hairtail quality is all starched with obtaining being with oppressing, and starch the shitosan of the 0.2-0.33% of quality with endo protease and the flesh of fish of the 0.36-0.97% of hairtail quality, stir 30-240min at 35-40 DEG C, pH is 5.5-8.5; After stirring terminates, regulate pH to 10-11.2, after keeping 30-45min, pH is adjusted to neutrality, then centrifugal under 4000rpm, get supernatant, hairtail sample solid will be obtained after supernatant bone dry, hairtail sample solid is added to refluxing extraction in Extraction solvent, centrifugal, get supernatant and be added on alkali alumina chromatographic column, collect post liquid, again with water elution, merged post liquid and water elution liquid, filtered, as need testing solution.
In the technical program, before hairtail sample extracts with Extraction solvent, first carry out endo protease enzymolysis to hairtail, making can faster by the constituents extraction of needs out in subsequent extracted technique, so that when efficient liquid phase chromatographic analysis, obtain data and finger-print more accurately.
As preferably, Extraction solvent is the one in absolute ethyl alcohol, acetone, ethyl acetate, chloroform.
As preferably, the chromatographic condition of high performance liquid chromatograph is: chromatographic column: AgilentEclipseXDBC18 post, 5 μm, 4.6mm × 250mm; Mobile phase: A phase is the butyl acetate of 0.05-0.07% or the ethylene glycol of 0.2-0.5%, and B phase is 0.2-0.5% potassium dihydrogen phosphate; Determined wavelength 210-320nm, column temperature is 38-40 DEG C, flow velocity: 1.1-1.5mL/min.
As preferably, endo protease is papain, Neutrase or Alcalase.
As preferably, the aperture of filtering in step a) is 0.22-0.43 μm.
As preferably, in step a), the solid-liquid ratio of hairtail sample and Extraction solvent is 1:50-150.
As preferably, the place of production of hairtail is Zhoushan, Dalian, Qingdao, ZhangZhou, the Indian Ocean.
Beneficial effect of the present invention:
1) the present invention utilizes HPLC chromatography to establish the finger-print of hairtail, and deemed-to-satisfy4 investigation shows that the method is reliable, repeatability is better, meets the requirement of finger-print;
2) the present invention detects that the method in the hairtail place of production is simple to operate, detection efficiency is high, testing result good reliability, fast, degree of accuracy is high, and accuracy rate is up to more than 92.7%.
Accompanying drawing explanation
Fig. 1 is the HPLC finger-print of embodiment 1 with a hairtail.
Fig. 2 is the HPLC finger-print of embodiment 2.
Fig. 3 is the cluster result of Different sources hairtail sample HPLC finger-print.
Embodiment
Below by way of specific embodiment, the present invention will be further explained:
In the present invention, if not refer in particular to, the raw material adopted all can be buied from market or this area is conventional, and the method in following embodiment, if no special instructions, is the conventional method of this area.
See the following form in the source building Different sources hairtail standard finger-print.
Table 1, Different sources hairtail sample source
| Sample number | The place of production | Source | Time |
| 1 | Zhoushan Zhoushan Fishing Ground | The industrial company limited in Zhejiang | In November, 2011 |
| 2 | Zhoushan Zhoushan Fishing Ground | Putuo, Zhoushan aquatic products company limited of Jinpeng | In November, 2011 |
| 3 | Zhoushan Zhoushan Fishing Ground | East River market, Zhoushan | In November, 2011 |
| 4 | Bohai Sea Gulf, Dalian | Dalian Li Yuan marine products company limited | In April, 2012 |
| 5 | The Qingdao Huanghai Sea | Jin Li source, Chengyang, Qingdao aquatic products firm | In Dec, 2011 |
| 6 | The Zhangzhou, Fujian East Sea | Dongshan County Xin Lian aquatic food company limited | Dec 3 in 2011--> |
| 7 | The Indian Ocean | Field, agriculture city, Hangzhou | In Dec, 2011 |
Embodiment 1
A construction method for hairtail finger-print, described method comprises:
A) preparation of need testing solution: hairtail sample choice body surface is rich in gloss, the cheek be scarlet, mucus is transparent, eyeball is full, cornea hairtail clearly, gets 1.5g hairtail muscle of back sample dry 12-18h at 0 to-10 DEG C, for subsequent use; Then the deionized water and stirring adding 5 times of hairtail quality is all starched with obtaining being with oppressing, and the shitosan of 0.2% of quality starched by the papain with 0.36% of hairtail quality and the flesh of fish, and stir 240min at 35 DEG C, pH is 5.5; After stirring terminates, regulate pH to 10, after keeping 30min, pH is adjusted to neutrality, then centrifugal under 4000rpm, get supernatant, hairtail sample solid will be obtained after supernatant bone dry, hairtail sample solid is added in ethyl acetate the ultrasonic extraction 40min that refluxes, centrifugal, getting supernatant is added on alkali alumina chromatographic column, collect post liquid, then with water elution, merge post liquid and water elution liquid, evaporate to dryness, dissolve with methyl alcohol and be settled to 20mL, then crossing 0.22 μm of miillpore filter, as need testing solution; Wherein, adopt gradient elution mode, elution program is in table 1;
B) high-performance liquid chromatogram determination: the need testing solution that aspiration step a) obtains measures to high performance liquid chromatograph, obtains the hairtail finger-print of Different sources; Wherein, the chromatographic condition of high performance liquid chromatograph is: chromatographic column: AgilentEclipseXDBC18 post, 5 μm, 4.6mm × 250mm; Mobile phase: A phase is the butyl acetate of 0.05%, B phase is 0.2% potassium dihydrogen phosphate; Determined wavelength 210nm, column temperature is 38 DEG C, flow velocity: 1.1mL/min, and sample size is 20 μ L;
C) determination of standard finger-print: the hairtail determining 5 Different sources totally 28 hairtail sample efficient liquid-phase chromatograph finger print atlas comparing, obtain the hairtail sample efficient liquid-phase chromatograph finger print atlas be made up of its total peak, there are 13 and 7 characteristic peaks in the common characteristic peak that this finger-print has.
Table 1, elution program table
| Time (min) | A phase | B phase |
| 0 | 5 | 95 |
| 5 | 12 | 90 |
| 10 | 29 | 73 |
| 16 | 36 | 67 |
| 24 | 59 | 51 |
| 31 | 83 | 16 |
| 41 | 89 | 10 |
| 54 | 97 | 4 |
| 60 | 100 | 0 |
Embodiment 2
A construction method for hairtail finger-print, described method comprises:
A) preparation of need testing solution: hairtail sample choice body surface is rich in gloss, the cheek be scarlet, mucus is transparent, eyeball is full, cornea hairtail clearly, gets 5g hairtail muscle of back sample dry 12-18h at 0 to-10 DEG C, for subsequent use; The hairtail sample crossing 55 mesh sieves is added in methyl alcohol the ultrasonic extraction 40min that refluxes, centrifugal, getting supernatant is added on alkali alumina chromatographic column, collect post liquid, then with water elution, merge post liquid and water elution liquid, evaporate to dryness, dissolve with methyl alcohol and be settled to 20mL, then crossing 0.35 μm of miillpore filter, as need testing solution; Wherein, adopt gradient elution mode, elution program is in table 1;
B) high-performance liquid chromatogram determination: the need testing solution that aspiration step a) obtains measures to high performance liquid chromatograph, obtains the hairtail finger-print of Different sources; Wherein, the chromatographic condition of high performance liquid chromatograph is: chromatographic column: AgilentEclipseXDBC18 post, 5 μm, 4.6mm × 250mm; Mobile phase: A phase is the butyl acetate of 0.06%, B phase is 0.2% potassium dihydrogen phosphate; Determined wavelength 210nm, column temperature is 40 DEG C, flow velocity: 1.3mL/min, and sample size is 20 μ L;
C) determination of standard finger-print: the hairtail determining 5 Different sources totally 28 hairtail sample efficient liquid-phase chromatograph finger print atlas comparing, obtain the hairtail sample efficient liquid-phase chromatograph finger print atlas be made up of its total peak, there are 13 and 7 characteristic peaks in the common characteristic peak that this finger-print has.
Embodiment 3
A construction method for hairtail finger-print, described method comprises:
A) preparation of need testing solution: hairtail sample choice body surface is rich in gloss, the cheek be scarlet, mucus is transparent, eyeball is full, cornea hairtail clearly, gets 4.5g hairtail muscle of back sample dry 12-18h at 0 to-10 DEG C, for subsequent use; Then the deionized water and stirring adding 6 times of hairtail quality is all starched with obtaining being with oppressing, and the shitosan of 0.33% of quality starched by the papain with 0.97% of hairtail quality and the flesh of fish, and stir 30min at 40 DEG C, pH is 7; After stirring terminates, regulate pH to 11.2, after keeping 45min, pH is adjusted to neutrality, then centrifugal under 4000rpm, get supernatant, hairtail sample solid will be obtained after supernatant bone dry, hairtail sample solid is added in ethyl acetate the ultrasonic extraction 40min that refluxes, centrifugal, getting supernatant is added on alkali alumina chromatographic column, collect post liquid, then with water elution, merge post liquid and water elution liquid, evaporate to dryness, dissolve with methyl alcohol and be settled to 20mL, then crossing 0.43 μm of miillpore filter, as need testing solution; Wherein, adopt gradient elution mode, elution program is in table 1;
B) high-performance liquid chromatogram determination: the need testing solution that aspiration step a) obtains measures to high performance liquid chromatograph, obtains the hairtail finger-print of Different sources; Wherein, the chromatographic condition of high performance liquid chromatograph is: chromatographic column: AgilentEclipseXDBC18 post, 5 μm, 4.6mm × 250mm; Mobile phase: A phase is the butyl acetate of 0.05%, B phase is 0.2% potassium dihydrogen phosphate; Determined wavelength 320nm, column temperature is 40 DEG C, flow velocity: 1.5mL/min, and sample size is 20 μ L;
C) determination of standard finger-print: the hairtail determining 5 Different sources totally 28 hairtail sample efficient liquid-phase chromatograph finger print atlas comparing, obtain the hairtail sample efficient liquid-phase chromatograph finger print atlas be made up of its total peak, there are 13 and 7 characteristic peaks in the common characteristic peak that this finger-print has.
According to the method for embodiment 1, get with a hairtail need testing solution, continuous sample introduction 5 times, measure HPLC finger-print (see figure 1), record chromatogram collection of illustrative plates overall picture directly perceived without significant difference, calculate relative reservation peak and the relative peak area value at each total peak, calculate RSD value, result display RSD value is all less than 5%, calculates similarity, the results are shown in Table 2 with " with similarity evaluation A version (2004) (Chinese Pharmacopoeia Commission) ".
Table 2, similarity test result
| Similarity | 1 | 2 | 3 | 4 | 5 | R |
| 1 | 1 | 0.989 | 0.996 | 0.989 | 0.990 | 0.998 |
| 2 | 0.989 | 1 | 0.989 | 0.996 | 0.989 | 0.996 |
| 3 | 0.996 | 0.989 | 1 | 0.989 | 0.995 | 0.998 |
| 4 | 0.989 | 0.996 | 0.989 | 1 | 0.989 | 0.996 |
| 5 | 0.990 | 0.989 | 0.995 | 0.989 | 1 | 0.960 |
| R | 0.998 | 0.996 | 0.998 | 0.996 | 0.960 | 1 |
As can be seen from Table 2,5 parts of hairtail test sample similarities are all greater than 0.90, illustrate that the precision of instrument is good.
The reference fingerprint collection of illustrative plates in each place of production comprises 13 mainly total peaks (accounting for the total area more than 90%) as can be seen from Figure 2, and the content that the place of production difference because of sample has peak has difference significantly.Wherein 5,6,8,9,11,12, No. 13 chromatographic peak degree of separation are good, and representativeness is comparatively strong, and fingerprint characteristic is obvious, as the characteristic peak of hairtail HPLC finger-print.No. 9 chromatographic peak degree of separation are better, peak height and peak area higher, retention time is suitable, as the reference peak of hairtail HPLC finger-print.
The relative peak area of table 3,28 Different sources hairtail characteristic peaks
| Relative peak area | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak |
| Numbering | 5 | 6 | 8 | 9 | 11 | 12 | 13 |
| S1 | 0.247 | 0.086 | 0.283 | 1.000 | 0.051 | 0.065 | 0.023 |
| S2 | 0.237 | 0.084 | 0.273 | 1.000 | 0.050 | 0.066 | 0.031 |
| S3 | 0.232 | 0.083 | 0.282 | 1.000 | 0.051 | 0.065 | 0.035 |
| S4 | 0.245 | 0.083 | 0.276 | 1.000 | 0.053 | 0.067 | 0.036 |
| S5 | 0.244 | 0.086 | 0.275 | 1.000 | 0.055 | 0.064 | 0.034 |
| S6 | 0.239 | 0.084 | 0.276 | 1.000 | 0.056 | 0.065 | 0.037 |
| S7 | 0.245 | 0.086 | 0.281 | 1.000 | 0.052 | 0.066 | 0.036 |
| S8 | 0.204 | 0.085 | 0.278 | 1.000 | 0.054 | 0.067 | 0.035 |
| S9 | 0.134 | 0.042 | 0.269 | 1.000 | 0.113 | 0.079 | 0.046 |
| S10 | 0.132 | 0.039 | 0.248 | 1.000 | 0.115 | 0.067 | 0.046 |
| S11 | 0.133 | 0.038 | 0.255 | 1.000 | 0.115 | 0.072 | 0.047 |
| S12 | 0.156 | 0.055 | 0.241 | 1.000 | 0.127 | 0.082 | 0.048 |
| S13 | 0.135 | 0.043 | 0.246 | 1.000 | 0.114 | 0.069 | 0.047 |
| S14 | 0.244 | 0.036 | 0.247 | 1.000 | 0.074 | 0.045 | 0.035 |
| S15 | 0.235 | 0.044 | 0.254 | 1.000 | 0.068 | 0.045 | 0.021 |
| S16 | 0.230 | 0.040 | 0.251 | 1.000 | 0.071 | 0.044 | 0.028 |
| S17 | 0.238 | 0.043 | 0.252 | 1.000 | 0.069 | 0.045 | 0.025 |
| S18 | 0.236 | 0.042 | 0.253 | 1.000 | 0.073 | 0.047 | 0.022 |
| S19 | 0.174 | 0.066 | 0.161 | 1.000 | 0.139 | 0.041 | 0.098 |
| S20 | 0.183 | 0.073 | 0.125 | 1.000 | 0.174 | 0.041 | 0.116 |
| S21 | 0.193 | 0.069 | 0.119 | 1.000 | 0.164 | 0.039 | 0.112 |
| S22 | 0.190 | 0.067 | 0.123 | 1.000 | 0.168 | 0.040 | 0.110 |
| S23 | 0.192 | 0.072 | 0.123 | 1.000 | 0.173 | 0.041 | 0.115 |
| S24 | 0.084 | 0.017 | 0.153 | 1.000 | 0.155 | 0.076 | 0.068 |
| S25 | 0.101 | 0.019 | 0.150 | 1.000 | 0.147 | 0.065 | 0.068 |
| S26 | 0.079 | 0.018 | 0.142 | 1.000 | 0.150 | 0.076 | 0.069 |
| S27 | 0.091 | 0.015 | 0.146 | 1.000 | 0.153 | 0.073 | 0.068 |
| S28 | 0.098 | 0.021 | 0.149 | 1.000 | 0.147 | 0.069 | 0.067 |
| Mean value | 0.184 | 0.055 | 0.219 | 1.000 | 0.105 | 0.060 | 0.054 |
The relative retention time of table 4,28 Different sources hairtail characteristic peaks
| Relative retention time | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak | Characteristic peak |
| Numbering | 5 | 6 | 8 | 9 | 11 | 12 | 13 |
| S1 | 0.815 | 0.891 | 0.929 | 1.000 | 1.064 | 1.085 | 1.145 6 --> |
| S2 | 0.812 | 0.889 | 0.928 | 1.000 | 1.065 | 1.086 | 1.147 |
| S3 | 0.810 | 0.887 | 0.928 | 1.000 | 1.066 | 1.087 | 1.147 |
| S4 | 0.812 | 0.891 | 0.929 | 1.000 | 1.065 | 1.085 | 1.146 |
| S5 | 0.814 | 0.891 | 0.929 | 1.000 | 1.064 | 1.085 | 1.145 |
| S6 | 0.813 | 0.889 | 0.928 | 1.000 | 1.065 | 1.086 | 1.146 |
| S7 | 0.814 | 0.890 | 0.929 | 1.000 | 1.065 | 1.085 | 1.145 |
| S8 | 0.812 | 0.891 | 0.928 | 1.000 | 1.064 | 1.085 | 1.146 |
| S9 | 0.818 | 0.893 | 0.931 | 1.000 | 1.064 | 1.084 | 1.142 |
| S10 | 0.818 | 0.893 | 0.931 | 1.000 | 1.064 | 1.084 | 1.142 |
| S11 | 0.817 | 0.819 | 0.893 | 1.000 | 1.065 | 1.085 | 1.142 |
| S12 | 0.810 | 0.888 | 0.927 | 1.000 | 1.066 | 1.088 | 1.151 |
| S13 | 0.816 | 0.892 | 0.930 | 1.000 | 1.065 | 1.0085 | 1.143 |
| S14 | 0.815 | 0.889 | 0.928 | 1.000 | 1.065 | 1.086 | 1.148 |
| S15 | 0.813 | 0.889 | 0.928 | 1.000 | 1.065 | 1.086 | 1.147 |
| S16 | 0.813 | 0.890 | 0.929 | 1.000 | 1.065 | 1.086 | 1.147 |
| S17 | 0.813 | 0.889 | 0.929 | 1.000 | 1.064 | 1.086 | 1.146 |
| S18 | 0.814 | 0.890 | 0.929 | 1.000 | 1.065 | 1.086 | 1.147 |
| S19 | 0.811 | 0.888 | 0.928 | 1.000 | 1.065 | 1.087 | 1.148 |
| S20 | 0.813 | 0.891 | 0.929 | 1.000 | 1.064 | 1.085 | 1.145 |
| S21 | 0.813 | 0.891 | 0.929 | 1.000 | 1.064 | 1.085 | 1.145 |
| S22 | 0.813 | 0.892 | 0.929 | 1.000 | 1.065 | 1.085 | 1.146 |
| S23 | 0.814 | 0.891 | 0.928 | 1.000 | 1.065 | 1.086 | 1.145 |
| S24 | 0.813 | 0.891 | 0.929 | 1.000 | 1.064 | 1.086 | 1.146 |
| S25 | 0.813 | 0.890 | 0.928 | 1.000 | 1.065 | 1.087 | 1.146 |
| S26 | 0.814 | 0.891 | 0.929 | 1.000 | 1.065 | 1.086 | 1.145 |
| S27 | 0.813 | 0.892 | 0.927 | 1.000 | 1.066 | 1.084 | 1.146 |
| S28 | 0.814 | 0.891 | 0.929 | 1.000 | 1.065 | 1.085 | 1.147 |
| Mean value | 0.814 | 0.888 | 0.927 | 1.000 | 1.065 | 1.083 | 1.146 |
As can be seen from Fig. 2, table 3, table 4, the relative retention time difference that the HPLC finger-print of 5 place of production hairtails has peak is less, and these 5 place of production hairtails are at least containing 13 kinds of above chemical compositions.But the relative peak area of each place of production hairtail also exists significant difference, the difference describing above 5 place of production hairtails living maritime space environment separately can cause the difference of principal ingredient and content.
In the hairtail finger-print of each place of production, using No. 9 chromatographic peaks as with reference to peak determine that its relative peak area is 1, the relative peak area at 13 total peaks in 28 batches of hairtail finger-prints is calculated with this, the relative peak area mainly having peak with these 13 is for index, hierarchial-cluster analysis is carried out with SPSS18.0 software, attached method between employing group, using square Euclidean distance estimating as sample similarity.The results are shown in Figure 3.
As can be seen from Fig. 3, it is a class that the hairtail sample in the same place of production gathers, and illustrates that between the same place of production, hairtail sample room chemical composition contamination difference is less; Between Different sources, clustering distance is different, and illustrates that between Different sources, the close and distant distant relationships of hairtail is different, comparatively close first the gathering of hydrological environment and geographical environment is a class, apart from nearlyer hairtail sample chemical composition and content more close.Hierarchial-cluster analysis can reflect the relation of sample room more comprehensively, all sidedly.
Claims (5)
1. a construction method for hairtail finger-print, is characterized in that, hairtail is prepared into need testing solution, detects through high performance liquid chromatograph, obtains the finger-print of hairtail;
Described method comprises the steps:
A) preparation of need testing solution: the hairtail sample getting Different sources, with Extraction solvent refluxing extraction, centrifugal, get supernatant to be added on alkali alumina chromatographic column, collect post liquid, then with water elution, merged post liquid and water elution liquid, filtered, as need testing solution;
B) high-performance liquid chromatogram determination: the need testing solution that aspiration step a) obtains measures to high performance liquid chromatograph, obtains the hairtail finger-print of Different sources; The chromatographic condition of high performance liquid chromatograph is: chromatographic column: AgilentEclipseXDBC18 post, 5 μm, 4.6mm × 250mm; Mobile phase: A phase is the butyl acetate of 0.05-0.07% or the ethylene glycol of 0.2-0.5%, and B phase is 0.2% potassium dihydrogen phosphate; Determined wavelength 210-320nm, column temperature is 38-40 DEG C, flow velocity: 1.1-1.5mL/min;
C) determination of standard finger-print: the hairtail determining 5 Different sources totally 28 hairtail sample efficient liquid-phase chromatograph finger print atlas comparing, obtain the hairtail sample efficient liquid-phase chromatograph finger print atlas be made up of its total peak, there are 13 and 7 characteristic peaks in the common characteristic peak that this finger-print has;
In step a) hairtail sample choice body surface be rich in gloss, the cheek be scarlet, mucus is transparent, eyeball is full, cornea hairtail clearly, gets 1.5-5.0g hairtail muscle of back sample dry 12-18h at 0 to-10 DEG C, for subsequent use; Then the deionized water and stirring adding 5-6 times of hairtail quality is all starched with obtaining being with oppressing, and starch the shitosan of the 0.2-0.33% of quality with endo protease and the flesh of fish of the 0.36-0.97% of hairtail quality, stir 30-240min at 35-40 DEG C, pH is 5.5-8.5; After stirring terminates, regulate pH to 10-11.2, after keeping 30-45min, pH is adjusted to neutrality, then centrifugal under 4000rpm, get supernatant, hairtail sample solid will be obtained after supernatant bone dry, hairtail sample solid is added to refluxing extraction in Extraction solvent, centrifugal, get supernatant and be added on alkali alumina chromatographic column, collect post liquid, again with water elution, merged post liquid and water elution liquid, filtered, as need testing solution; Wherein, the aperture of filtration is 0.22-0.43 μm;
Wherein, mobile phase elution program is:
Time (min) A phase B phase
0595
51290
102973
163667
245951
318316
418910
54974
601000。
2. the construction method of a kind of hairtail finger-print according to claim 1, is characterized in that, Extraction solvent is the one in absolute ethyl alcohol, acetone, ethyl acetate, chloroform.
3. the construction method of a kind of hairtail finger-print according to claim 1, is characterized in that, endo protease is papain, Neutrase or Alcalase.
4. the construction method of a kind of hairtail finger-print according to claim 1, is characterized in that, in step a), the solid-liquid ratio of hairtail sample and Extraction solvent is 1:50-150.
5., according to the construction method of claim 1 or 3 or 4 or described a kind of hairtail finger-print, it is characterized in that, the place of production of hairtail is Zhoushan, Dalian, Qingdao, ZhangZhou, the Indian Ocean.
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| FTIR光谱法对带鱼贮藏品质的研究;孟志娟 等;《食品科技》;20131231;第38卷(第2期);280-284 * |
| 基于多元指纹图谱技术的带鱼产地鉴别与新鲜度评价研究;孟志娟;《中国优秀硕士学位论文全文数据库 农业科技辑》;20131015(第10期);第二章第1.2.2、3.2.2、3.2.3.6、3.2.4.3、3.3.2小节 * |
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