CN104897821A - Method and kit for extracting inositol in sample, and application of kit - Google Patents
Method and kit for extracting inositol in sample, and application of kit Download PDFInfo
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- CN104897821A CN104897821A CN201510297053.9A CN201510297053A CN104897821A CN 104897821 A CN104897821 A CN 104897821A CN 201510297053 A CN201510297053 A CN 201510297053A CN 104897821 A CN104897821 A CN 104897821A
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Abstract
The invention belongs to the technical field of analysis, and relates to a method for extracting inositol in a sample. The method comprises the step of soaking the sample by using an ammonium acetate water solution. The invention further relates to a kit for extracting the inositol in the sample. The kit comprises the ammonium acetate water solution, and an aqueous phase filter membrane. The kit is used for extracting the inositol from tobaccos or tobacco products, or used for quality control of the tobaccos or the tobacco products.
Description
Technical field
The invention belongs to analysis technical field, relate to a kind of method, kit and uses thereof of extracting sample mysoinositol.
Background technology
Inositol (Inositol), also known as 1,2,3,4,5,6-inositol, be separated and obtain from cardiac muscle and liver the earliest, molecular formula is C
6h
12o
6, structure is such as formula shown in I.
Inositol is extensively distributed in animal and plant body, is to participate in metabolic activity " bios ", has the effect of immunity, some disease of prevention and therapy.If higher mammal lacks inositol, will there will be the phenomenon such as growth retardation and trichomadesis, the demand of human body to inositol is 1-2g/ days, and many health drinks and infant foods are all added with micro-inositol.The growth factor of certain micro-organisms in inositol or intestines, when other hypovitaminosis, the vitamin that it can stimulate Microbe synthesis to lack.In fermentation and food industry, inositol can be used for multi-cultur es and cultivates, promotes that yeast increases.In addition, the inositol content in tobacco is higher, between 0.1% ~ 1%, and its inositol content of the tobacco leaf of different regions and kind slightly difference.
At present, the detection method for inositol mainly contains gravimetric method, microbial method, potassium metaperiodate oxidizing process, differential refraction method, high performance liquid chromatography and vapor-phase chromatography etc.Wherein, the easy contaminated environment of gravimetric method; Potassium metaperiodate oxidizing process can not get rid of the interference of vicinal diol compound, specificity is poor; Differential pulse polarograpll method instrument is unstable, disturbing factor is many; High performance liquid chromatography is for complicated component sample separation weak effect.And, all there is the problems such as extraction step is loaded down with trivial details, the time is longer in the pre-treatment of current inositol detection method, especially because inositol boiling point is high, adopts during vapor-phase chromatography and need carry out pre-column derivatization, just can measure after being prepared into low boiling ester class, pre-treatment is comparatively complicated.
To still need at present a kind of easy pre-treating method, take out the inositol of sample especially in tobacco or tobacco product with Quick.
Summary of the invention
The present inventor obtains a kind of method of rapid extraction sample mysoinositol, is particularly useful for extracting the higher tobacco of inositol content or tobacco product, and the method is fast and convenient, selectivity good, favorable reproducibility.In addition, inventor also adopts Liquid Chromatography-Mass Spectrometry to detect the inositol in extract first, highly sensitive.
First aspect present invention relates to a kind of method extracting sample mysoinositol, comprises the steps: to infiltrate sample with ammonium acetate solution, obtains extract.
When described infiltration refers to that ammonium acetate solution and sample come in contact, liquid is attached to sample surfaces or penetrates into the phenomenon of sample interior.Particularly, during infiltration, ammonium acetate solution liquid level is at least 1/3 of height of specimen or be at least 1/2 of height of specimen, preferably, and the complete submergence sample of ammonium acetate solution.
In the method for any one of first aspect present invention, described sample is tobacco or tobacco product.
Inventor finds, the selection of Extraction solvent has appreciable impact to inositol analysis, and ammonium acetate solution can by the inositol rapid extraction in sample out, and extraction ratio is high and method is simple.And adopt other buffer solution of sodium phosphate (NaH
2pO
4-Na
2hPO
4), buffer solution of potassium phosphate (K
2hPO
4-KH
2pO
4) etc. cannot carry out effective extraction and analysis to the inositol in sample.
In the method for any one of first aspect present invention, the concentration of described ammonium acetate solution is 0.09-0.15mol/L, is specially 0.1-0.15mol/L or 0.12-0.15mol/L, is preferably 0.1mol/L.
The concentration of ammonium acetate solution is 0.09-0.15mol/L, can extract the inositol in sample substantially completely.
In the method for any one of first aspect present invention, the amount of adding ammonium acetate solution in sample every gram described is 250-1000mL, is specially 250mL, 500mL, 750mL or 1000mL, is preferably 500mL/g.
Inventor finds, when ammonium acetate solution addition is excessive, weak output signal, easily by the impact of baseline noise; When addition is too small, for the sample that inositol content is high, easily there is signal value supersaturation, measure inaccurate problem.Control at 250-1000mL/g by ammonium acetate solution addition, especially 500mL/g, can possess normal signal value, make measurement accurate, suitable ammonium acetate solution addition can make inositol evenly be extracted simultaneously.
In the method for any one of first aspect present invention, infiltrating time >=10min, particularly, infiltrating time >=30min, >=40min or >=50min, be preferably 30min.
In the method for any one of first aspect present invention, described infiltration is carried out under ultrasonic; Particularly, ultrasonic power is 40% of peak power; More specifically, infiltration temperature is 30-60 DEG C, is specially 30 DEG C, 40 DEG C, 50 DEG C or 60 DEG C.
In the method for any one of first aspect present invention, also comprise the step of being carried out by extract filtering; Particularly, with aqueous phase membrane filtration extract; More specifically, the aperture of described aqueous phase filter membrane is 0.22 μm.
In the method for any one of first aspect present invention, also comprise the step adopting LC-MS analysis extract.
The method arbitrary according to the present invention, liquid phase chromatogram condition is any one in following (1)-(6) or multinomial:
(1) chromatographic column is Féraud door C
18liquid-phase chromatographic column;
(2) sample size is 5 μ L;
(3) mobile phase is 0.1% aqueous acetic acid;
(4) flow velocity is 0.8mL/min;
(5) column temperature is 40 DEG C;
(6) isocratic elution program is adopted, elution time 3min.
Inventor finds to adopt Féraud door C
18liquid-phase chromatographic column analyzes extract, and the chromatographic peak type obtained is better, response is higher; Chromatographic peak response increases along with sample size and increases, but when inventor finds that sample size is too high, crack appears in inositol chromatographic peak, have impact on the accuracy of detection; Make mobile phase with 0.1% aqueous acetic acid, chromatographic peak better, response is higher.
The method arbitrary according to the present invention, Mass Spectrometry Conditions is any one in following (1)-(7) or multinomial:
(1) scan mode is negative ion scanning;
(2) quota ion is to 179/87, qualitative ion pair 179/59;
(3) impact energy is-25V;
(4) ion gun is electron spray ionisation source;
(5) detection mode is many reaction detection;
(6) electron spray voltage is-4500V;
(7) ion source temperature is 550 DEG C.
With negative-ion mode scanning, impact energy be-25V, and selection quota ion is to 179/87, and qualitative ion pair 179/59 detects, and accuracy is high, highly sensitive, the recovery is high, reproducible.Select other impact energies and quota ion lower to accuracy during detection.
Second aspect present invention relates to a kind of kit extracting sample mysoinositol, comprises ammonium acetate solution and aqueous phase filter membrane.
The kit that second aspect present invention is arbitrary, to comprise in following (1)-(5) arbitrary one or multinomial:
(1) concentration of described ammonium acetate solution is 0.09-0.15mol/L, is preferably 0.1mol/L;
(2) amount of ammonium acetate solution is at least 250mL, is specially 250-1000mL, is preferably 500mL;
(3) aperture of described aqueous phase filter membrane is 0.22 μm;
(4) described kit is quantification assay kit;
(5) described sample is tobacco or tobacco product.
Third aspect present invention relates to described kit at extraction sample mysoinositol, or the purposes in Quality control quality; Particularly, described sample is tobacco or tobacco product.
The beneficial effect of the invention
(1) the present invention infiltrates sample with ammonium acetate solution, can extract inositol from sample particularly tobacco or tobacco product, and method is simple, without the need to using virose organic solvent.
(2) the present invention adopts Liquid Chromatography-Mass Spectrometry to analyze inositol in extract first, quick and precisely, highly sensitive, simple to operate.
(3) use the recovery of the inventive method extraction sample mysoinositol high, reproducible.When detecting the sample of tobacco or tobacco product, there is assorted peak hardly, object linear dependence is better, standard solution is diluted certain multiple, calculate detecting of inositol with signal to noise ratio (S/N ratio) S/N=3 and be limited to 0.07mg/g, what calculate inositol with signal to noise ratio (S/N ratio) S/N=10 is quantitatively limited to 0.22mg/g, and average recovery rate is 93.4% ~ 94.9%, relative standard deviation RSD<5% (n=6).
Accompanying drawing explanation
Fig. 1: the process flow diagram of analytical approach in the embodiment of the present invention 1.
Fig. 2: the chromatogram of the embodiment of the present invention 1 Plays solution.
Fig. 3: the chromatogram of extract in the embodiment of the present invention 1.
Fig. 4: the mass spectrogram of the embodiment of the present invention 1 mysoinositol.
Fig. 5: the canonical plotting of the embodiment of the present invention 1 mysoinositol.
Fig. 6: the graph of a relation of different solvents and chromatographic peak area in the embodiment of the present invention 2.
Fig. 7: chromatogram when addition is 25mL ammonium acetate solution in the embodiment of the present invention 3;
Fig. 8: chromatogram when addition is 100mL ammonium acetate solution in the embodiment of the present invention 3;
Fig. 9: the graph of a relation of different extraction time and the inositol content recorded in the embodiment of the present invention 4.
Figure 10: in the embodiment of the present invention 7, pure water makes the chromatogram of mobile phase.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
embodiment 1: to tobacco product mysoinositol containing quantitative analysis
Analytical approach is as shown in Figure 1:
(1) preparing standard solution: take 0.1g inositol, be accurate to 0.0001g, is transferred to constant volume in the brown volumetric flask of 100mL after dissolving, is mixed with 1mg/mL standard reserving solution with 0.1mol/L ammonium acetate solution; Pipette the standard reserving solution of 0.1mL, 0.5mL, 1mL, 2mL, 3mL and 5mL respectively in 100mL volumetric flask, with 0.1mol/L ammonium acetate solution constant volume, obtain the standard solution that inositol concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL and 50 μ g/mL, lucifuge 4 DEG C preservation, under being positioned over normal temperature in advance when taking, can use after reaching normal temperature.Do weekly a graticule.
(2) extract of sample is prepared: take about 0.1g pipe tobacco, be placed in 150mL ground triangular flask, accurately add the ammonium acetate solution of 50mL, 0.1mol/L, ultrasonic extraction 30min, ultrasonic power is 40%, after 0.22 μm of aqueous phase membrane filtration, obtain extract.
(3) LC-MS/MS method is adopted to analyze extract, standard solution respectively.
Chromatographiccondition: adopt Féraud door C
18liquid-phase chromatographic column, column temperature is 40 DEG C; Sample size 5 μ L; Mobile phase is 0.1% aqueous acetic acid, and flow velocity is 0.8ml/min; Adopt isocratic elution program, elution time is 3min altogether.
As shown in Figure 2, the stratographic analysis result of extract as shown in Figure 3 for the stratographic analysis result of standard solution.
Mass spectrophotometry condition: ion gun: electron spray ionisation source (ESI); Scan mode: negative ion scans; Detection mode: multiple-reaction monitoring (MRM); Electron spray voltage :-4500V; Ion source temperature: 550 DEG C; Assisted gas Gas1 pressure: 60psi; Assisted gas Gas2 pressure: 50psi; Remove a bunch voltage (DP) :-40V; Impact energy (CE) :-25V; Ion pair select: quota ion to 179/87, qualitative ion pair 179/59.The mass spectrogram of extract as shown in Figure 4.
(4) result of calculation:
Carry out regretional analysis with the chromatographic peak area of standard solution mysoinositol to its respective concentration, obtain typical curve as shown in Figure 5, concrete data are in table 1.
Table 1
By the chromatographic peak area of extract chromatogram mysoinositol, substitute into above typical curve, obtain the inositol content in extract, calculate the inositol content in pipe tobacco according to the following formula.
m＝1000×(A×S)/n
In formula:
Inositol content (mg/g) in m-pipe tobacco;
Inositol content (μ g/mL) in A-extract;
S-extracting liquid volume (mL);
The quality (g) of n-take pipe tobacco.
The inositol content of the present embodiment pipe tobacco is 9.3mg/g.
From table 1 and Fig. 4, assorted peak is there is hardly in the chromatographic condition adopted in tobacco product actual sample is analyzed, object has good linear dependence, and mixed standard solution is diluted certain multiple, calculates detecting of inositol be limited to 0.07mg/g with signal to noise ratio (S/N ratio) S/N=3.
embodiment 2: the comparison of different solvents and variable concentrations
The present embodiment uses pure water, buffer solution of sodium phosphate (NaH respectively
2pO
4-Na
2hPO
4), buffer solution of potassium phosphate (K
2hPO
4-KH
2pO
4) and the ammonium acetate solution of 0.03mol/L, 0.09mol/L, 0.12mol/L, 0.15mol/L tobacco product is extracted, other conditioned reference embodiments 1.
Buffer solution of sodium phosphate, buffer solution of potassium phosphate extract tobacco product as Extraction solvent and chromatographic peak do not detected.The peak area of other Extraction solvent stratographic analyses as shown in Figure 6.
As shown in Figure 6, compared with ammonium acetate solution, the inositol extraction ratio of Aqua pure extract tobacco product is low.As ammonium acetate solution concentration >=0.1mol/L, the extraction ratio of inositol reaches stable substantially, illustrates that the inositol of tobacco product can extract by 0.1mol/L ammonium acetate solution substantially completely.
embodiment 3: the comparison of ammonium acetate solution Different adding amount
The present embodiment is respectively to the 0.1mol/L ammonium acetate solution adding 25ml, 50ml, 75ml and 100ml in tobacco product, and other reference examples 1, the results are shown in Table 2.
Table 2
As shown in Table 2, for different ammonium acetate solution additions, result no significant difference.As Figure 7-8, when addition is larger, as the 100mL addition of Fig. 8, signal value is less, is subject to baseline noise impact, and when addition is less, as the 25mL addition of Fig. 7, easily occurs signal value supersaturation, affect measurement result.In order to the accuracy of the completeness and detection of taking into account extraction, ensure the homogeneity that samples simultaneously and reduce reagent dosage, experimental selection addition is 50mL.
embodiment 4: the comparison of different extraction time
The present embodiment is respectively using 10min, 20min, 30min, 40min and 50min as extraction time, and the inositol content recorded separately as shown in Figure 9.As ultrasonic time >=30min, the inositol content recorded is basicly stable, and the extraction ratio of inositol is the highest.For avoiding the ultrasonic temperature of sample that causes for a long time significantly to raise, simultaneously in order to make the inositol in different sample be fully extracted out, extraction time adopts 30min.
embodiment 5: the comparison of different Mass Spectrometry Conditions
MS/MS collision (CE) can be set as-10V by the present embodiment, quota ion to being 179/161, other reference examples 1.The extract chromatographic peak area obtained is only 34% of embodiment 1 extract chromatographic peak area.
embodiment 6: the comparison of different chromatographic column
The present embodiment is respectively with chromatographic column ZORBAX Eclipse C18 (100mm × 2.1mm that Agilent company produces, 1.8 μm), ZORBAX Eclipse XDB-C8 (150mm × 4.6mm, 5m), ZORBAX SB-C3 (150mm × 2.1mm, 5 μm), ZORBAX SB-C3 (100mm × 3.0mm, 1.8 μm), ZORBAX XDB-Phenyl (150mm × 4.6mm, 3.5 μm), ZORBAX 300-SCX (150mm × 2.1mm, 5 μm), power & light company produce chromatographic column APS-2Hypersil (100mm × 2.1mm, 5 μm) and Féraud door Luna 5u C18 chromatographic column (150.0mm × 3.9mm, 5 μm) carry out analysis mensuration, other reference examples 1, result shows, Féraud door Luna 5u C18 chromatographic column (150.0mm × 3.9mm, 5 μm) chromatographic peak type best, response is the highest.
embodiment 7: the comparison of different mobile phase
The present embodiment is analyzed using pure water as mobile phase, and other reference examples 1, obtain chromatogram as shown in Figure 10.
By more known for Figure 10 and Fig. 2-3, when making mobile phase with pure water, the peak intensity of inositol is lower, and when adopting 0.1% acetic acid as mobile phase, chromatographic peak type is better, and response is higher.
embodiment 8: the comparison of different sample size
The present embodiment respectively with 1 μ L, 2 μ L and 10 μ L sample introductions, other reference examples 1.
Result shows, chromatographic peak response can increase along with the increase of sample size, and when reaching the sample size of 10 μ L, inositol chromatographic peak occurs splitting peak.
embodiment 9: the investigation of repeatability and recovery of standard addition
Respectively to the standard solution adding 5mg/g, 10mg/g and 20mg/g tri-kinds of variable concentrations in sample, carry out recovery of standard addition test, each sample measures 6 times respectively, analytical approach reference example 1.According to the recovery of standard addition of Analysis result calculation tobacco product mysoinositol, result is as shown in table 3.
The recovery of table 3 tobacco product mysoinositol and repeatability (n=6)
As can be seen from Table 3, in 3 mark-on levels, utilize the method analysis to detect the recovery of tobacco product mysoinositol between 93.4%-94.9%, the relative standard deviation of sample tests is less than 5%, illustrates that the recovery of this law is higher, and repeatability better.
embodiment 10: the mensuration of different tobacco product inositol content
Adopt the inositol content in the analytical approach mensuration Different sources of embodiment 1, dissimilar tobacco leaf, the results are shown in Table 4.
The inositol content of table 4 Different sources, dissimilar tobacco leaf
| Sample | Inositol content (mg/g) |
| Domestic flue-cured tobacco 1 | 9.3 |
| Domestic flue-cured tobacco 2 | 7.33 |
| Domestic flue-cured tobacco 3 | 5.87 |
| Domestic flue-cured tobacco 4 | 6.13 |
| Domestic flue-cured tobacco 5 | 8.91 |
| Domestic flue-cured tobacco 6 | 8.76 |
| External tobacco leaf 1 | 8.11 |
| External tobacco leaf 2 | 5.77 |
| External tobacco leaf 3 | 5.67 |
| Burley tobaccos 1 | 0.64 |
| Burley tobaccos 2 | 0.73 |
| Burley tobaccos 3 | 0.88 |
| Turkish tobaccos 1 | 4.85 |
| Turkish tobaccos 2 | 5.96 |
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (10)
1. extract a method for sample mysoinositol, comprise the steps:
Infiltrate sample with ammonium acetate solution, obtain extract.
2. method according to claim 1, wherein, described sample is tobacco or tobacco product.
3. method according to claim 1, wherein, the concentration of described ammonium acetate solution is 0.09-0.15mol/L, is preferably 0.1mol/L.
4. method according to claim 1, wherein, the amount of adding ammonium acetate solution in sample every gram described is 250-1000mL, is preferably 500mL.
5. method according to claim 1, wherein, infiltrating time >=10min, particularly, infiltrating time >=30min, is preferably 30min.
6. method according to claim 1, wherein, described infiltration is carried out under ultrasonic; More specifically, infiltration temperature is 30-60 DEG C.
7. method according to claim 1, wherein, also comprises the step of being carried out by extract filtering; Particularly, with aqueous phase membrane filtration extract; More specifically, the aperture of described aqueous phase filter membrane is 0.22 μm.
8. extract a kit for sample mysoinositol, comprise ammonium acetate solution and aqueous phase filter membrane.
9. kit according to claim 8, to is characterized in that in following (1)-(5) arbitrary one or multinomial:
(1) concentration of described ammonium acetate solution is 0.09-0.15mol/L, is preferably 0.1mol/L;
(2) amount of ammonium acetate solution is at least 250mL, is specially 250-1000mL, is preferably 500mL;
(3) aperture of described aqueous phase filter membrane is 0.22 μm;
(4) described kit is quantification assay kit;
(5) described sample is tobacco or tobacco product.
10. kit described in claim 8 or 9 is extracting the inositol in sample, or the purposes in Quality control quality; Particularly, described sample is tobacco or tobacco product.
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