CN109270186A - A kind of Dan peach kernel formulation characteristics map detection method - Google Patents
A kind of Dan peach kernel formulation characteristics map detection method Download PDFInfo
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Abstract
The present invention provides a kind of characteristic spectrum detection methods of Dan peach kernel preparation, it is characterised in that: it is detected using high performance liquid chromatography, includes the following steps: 1) to prepare reference solution；2) test solution is prepared；3) test sample to be measured is taken, is detected using high performance liquid chromatography；4) characteristic spectrum is analyzed, 5 characteristic peaks should be presented in test sample characteristic spectrum, wherein 2 peaks should be identical as corresponding object of reference peak retention time respectively, peak corresponding with amarogentin object of reference is the peak S, peak corresponding with tryptophan object of reference is peak 2, the relative retention time of each characteristic peak Yu the peak S is calculated, relative retention time should be within ± the 8% of specified value.The characteristic spectrum detection method of Dan peach kernel preparation of the invention, it can be used for the HPLC characteristic spectrum of Dan peach kernel medicinal material-medicine materical crude slice-extract and granule, Dan peach kernel medicinal material-medicine materical crude slice-extract and the multicomponent whole looks of granule can be reflected, provide new method for the control of the Dan peach kernel quality of the pharmaceutical preparations.
Description
Technical field
Present invention relates particularly to a kind of Dan peach kernel formulation characteristics map detection methods.
Background technique
Peach kernel (Semem Persicae) also known as Semen Persicae are rosaceous plant peach Prunus persica (L.)
The dry mature seed of Bastsch or mountain peach Prunusdavidiana (Carr.) Franch.Peach all parts of the country are generally planted,
It is picked when fruit maturation in 6~July, peach is removed into pulp and nucleocapsid, seed is taken out, dries, that is, be used for medicinal purpose.Peach kernel is sought there are many containing
It forms point and bioactive substance, is the significant natural drug of clinical efficacy.There are also certain antibacterials for its water decoction and extract
Analgesia, antiallergy, it is antitumor the effects of.
The difference of peach kernel and Dan peach kernel is mainly: peach kernel is to take crude drug, removes impurity and remaining hard shell, weeds out ash
Bits, used time smash to pieces.Dan peach kernel is that peach kernel is taken to set in boiling water, and heating, which is boiled to micro- swell of kind of skin, to be pulled out, is slightly steeped in cold water, is fished out
It rises, kind of skin and kernel is rubbed open, dry, kind of a skin of winnowing with a dustpan, the used time smashs to pieces.In addition to this, the two is essentially identical, can be used for: the moon
Through uncomfortable, amenorrhoea , lumps in the chest and abdomen, postpartum stasis abdominal pain.
Main chemical compositions in peach kernel and Dan peach kernel are amygdalin, are mostly at present to survey to the chemical constitution study of peach kernel
Fixed wherein Glucoside Content.High performance liquid chromatography (HPLC) is to measure one of the common method of amygdalin in peach kernel, also
There are ultraviolet spectrophotometry, volumetric method, Second derivative spectroscopy direct measuring method, thin layer chromatography scanning, fluorescence quemching method etc..These
Method differs from one another, but is all to measure Glucoside Content as sole purpose, and the chemical component of peach kernel or Dan peach kernel is more multiple
Miscellaneous, the quality of peach kernel or Dan peach kernel cannot comprehensively be reflected by only only surveying amygdalin therein.Due to peach kernel and Dan peach kernel
The two is essentially identical, and existing research has concentrated on peach kernel, has not been reported to the research of Dan peach kernel.
Summary of the invention
To solve the above problems, the present invention provides a kind of characteristic spectrum detection method of Dan peach kernel preparation, feature exists
In: it is detected using high performance liquid chromatography, and operating procedure is as follows:
1) preparation of reference solution: taking amarogentin, tryptophan product, and respectively plus methanol dissolves, as object of reference
Solution；
2) preparation of test solution:
A medicinal material or medicine materical crude slice test sample: taking test sample, extracting in water, and filtering takes filtrate to get test solution；
B standard decoction or granule test sample: taking test sample, and 70% methanol is added to extract, and filtering takes filtrate, must both supply
Test sample solution；
3) reference solution being drawn respectively and test solution injecting liquid chromatograph, chromatographic condition is as follows: chromatographic column: with
Octadecylsilane chemically bonded silica is filler；Mobile phase: using methanol as mobile phase A, using 0.1% phosphoric acid solution as Mobile phase B
Gradient elution；Gradient elution program is as follows:
4) characteristic spectrum is analyzed.
Further, step 1) the reference substance amarogentin, Tryptophan concentration are respectively every 1ml containing 1mg, 0.01mg
Solution.
Further, the mass volume ratio of medicinal material or medicine materical crude slice and water described in step a is 1g:50mL.
Further, it is extracted as decocting described in step a and extract, decoction boiling time is 30min.
Further, the mass volume ratio of standard decoction or granule and 70% methanol described in step b is 1g:125mL.
Further, ultrasonic extraction, ultrasonic power 600W, frequency 40kHz, time 30min are extracted as described in step b.
Further, the reference solution and test solution amount that liquid chromatograph is injected separately into described in step 3) are 10
~20 μ l, preferably 10 μ l.
Further, the wavelength of the step 3) chromatographic condition is 214nm, and column temperature is 25 DEG C, and flow rate of mobile phase is
1.00ml/min, number of theoretical plate is calculated by amarogentin peak should be not less than 3000.
Further, step 3) the octadecylsilane chemically bonded silica chromatographic column is 5 μm 4.6 of Agilent HC-C18
×250mm、phenomenex Lna 5μm C18(2)100A 250×4.6mm 5micron、Diamonsil C18(2)5μm
250 × 4.6mm, preferably 5 μm of 4.6 × 250mm of Agilent HC-C18.
Further, 5 characteristic peaks should be presented in the characteristic spectrum of the step 4), wherein 2 peaks should respectively and accordingly
Object of reference peak retention time it is identical, peak corresponding with amarogentin object of reference be the peak S, peak corresponding with tryptophan object of reference is
Peak 2, calculates the relative retention time of each characteristic peak Yu the peak S, and relative retention time should be within ± the 8% of specified value.Regulation
Value are as follows: 0.880 (peak 1), 0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
Dan peach kernel formulation characteristics map detection method of the invention, can detect amygdalin, tryptophan simultaneously, can be used for
The quality testing of Dan peach kernel medicinal material-medicine materical crude slice-extract and granule can reflect Dan peach kernel medicinal material-medicine materical crude slice-extract and match
As a result reliably the multicomponent whole looks of square particle provide new method, Dan peach kernel medicine for the quality control of Dan peach kernel particle
Material, medicine materical crude slice, extract, have a good application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 Dan peach kernel medicinal material characteristic spectrum (S1:010439-1611001；S2:010439-1611002；S3:010439-
1611003；S4:010439-1611004；S5:010439-1612001；S6:010439-1612002；S7:XLS1802115；
S8:XLS1802116；S9:XLS1802117；S10:XLS1802118；S11:XLS1802119；S12:XLS1802158；S13:
XLS1802159；S14:XLS1802160；S15:XLS1802160；S16:XLS1802162)
Fig. 2 Dan peach kernel medicine materical crude slice characteristic spectrum (S1:DTR180201；S2:DTR180202；S3:DTR180203；S4:
DTR180204；S5:DTR180205；S6:DTR180206；S7:DTR180207；S8:DTR180208；S9:DTR180209；
S10:DTR1802010；S11:DTR1802011；S12:DTR1802012；S13:DTR1802013；S14:DTR1802014；
S15:DTR180215, S16:DTR180216)
Fig. 3 Dan peach kernel standard decoction characteristic spectrum (S1:DTRBT180201；S2:DTRBT180202；S3:
DTRBT180203；S4:DTRBT180204；S5:DTRBT180205；S6:DTRBT180206；S7:DTRBT180207；S8:
DTRBT180208；S9:DTRBT180209；S10:DTRBT1802010；S11:DTRBT1802011；S12:
DTRBT1802012；S13:DTRBT1802013；S14:DTRBT1802014；S15:DTRBT180215, S16:
DTRBT180216)
Fig. 4 Dan peach kernel granule characteristic spectrum (S1:SY1804001；S2:SY1804002；S3:SY1804003；S4:
SY1804004；S4: peach kernel control medicinal material)
Fig. 5 medicinal material, medicine materical crude slice, standard decoction, Dan peach kernel granule compare map stacking chart (peak 2: tryptophan；Peak 3 (S):
Amarogentin)
Fig. 6 Dan peach kernel granule specificity experimental comparison figure
Fig. 7 Waters chromatograph investigates map
Fig. 8 Shimadzu 20AT chromatograph investigates map
1260 type chromatograph of Fig. 9 Agilent investigates map
Figure 10 Dan peach kernel standard decoction chromatographic column durability investigates map
Figure 11 Dan peach kernel granule characteristic spectrum
Figure 12 Dan peach kernel granule compare feature map
Specific embodiment
1, material, reagent and instrument
1.1 laboratory apparatus
High performance liquid chromatograph: 1260 type high performance liquid chromatograph of Agilent；Electronic balance: ME204E/02, MS205DU,
XP26 (plum Teller-support benefit Instrument Ltd.)；Ultrapure water machine: cellular type 1810A (the limited public affairs of Shanghai Moller scientific instrument
Department)；Ultrasonic cleaner: KQ5200DB type (600W, 40KHz；Kunshan Ultrasonic Instruments Co., Ltd.)；Chromatographic column: Agilent
5 μm of C18 (2) 250 × 4.6mm of 100A 5micron of (2) 4.6 × 250mm of 5HC-C18 (Shimadzu), phenomenex Lna
(2) 5 μm of 250 × 4.6mm (enlightening horse) of (Féraud door), Diamonsil C18.
1.2 reagents and reagent
Acetonitrile, methanol, phosphoric acid are chromatographically pure, and water is ultrapure water；
Amarogentin (National Institute for Food and Drugs Control, lot number: 110820-201607, content is in terms of 90.7%)；
Tryptophan (National Institute for Food and Drugs Control, lot number: 140686-201303, content is in terms of 99.9%)；
Peach kernel control medicinal material (National Institute for Food and Drugs Control, lot number: 120953-201407)；
Dan peach kernel granule (preparation of Sichuan new green medicine company development in science and technology Co., Ltd, lot number: SY1804001,
SY1804002,SY1804003)；
Dan peach kernel medicinal material (Sichuan new green medicine company development in science and technology Co., Ltd, lot number: 010439-1611001,010439-
1611002、010439-1611003、010439-1611004、010439-1612001、010439-1612002、
XLS1802115、XLS1802116、XLS1802117、XLS1802118、XLS1802119、XLS1802158、XLS1802159、
XLS1802160,XLS1802160,XLS1802162)；
Dan peach kernel medicine materical crude slice (Sichuan new green medicine company development in science and technology Co., Ltd, lot number: DTR180201, DTR180202,
DTR180203、DTR180204、DTR180205、DTR180206、DTR180207、DTR180208、DTR180209、
DTR1802010,DTR1802011,DTR1802012,DTR1802013,DTR1802014,DTR180215,DTR180216)；
Dan peach kernel standard decoction (preparation of Sichuan new green medicine company development in science and technology Co., Ltd, lot number: DTRBT180201,
DTRBT180202、DTRBT180203、DTRBT180204、DTRBT180205、DTRBT180206、DTRBT180207、
DTRBT180208、DTRBT180209、DTRBT1802010、DTRBT1802011、DTRBT1802012、DTRBT1802013、
DTRBT1802014、DTRBT180215、DTRBT180216)。
1 Dan peach kernel medicinal material characteristic spectrum of embodiment
1) preparation of reference solution:
Amarogentin, tryptophan product are taken, respectively plus every 1ml amarogentin containing 1mg, 0.01mg tryptophan is made in methanol
Reference solution；
2) preparation of test solution:
16 crowdes of Dan peach kernel medicinal material 1g are taken respectively, and accurately weighed, accurate respectively that water 50ml is added, weighed weight boils 30 points
Clock is let cool, then weighed weight, and the weight of less loss is supplied with water, is shaken up, filtration, as test solution；
3) measurement of characteristic spectrum:
It is accurate respectively to draw reference solution and 10 μ l of test solution, liquid chromatograph is injected, chromatogram is obtained；
Chromatographic condition is as follows:
Detection wavelength: 214nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: methanol (A) and 0.1% phosphoric acid solution (B), gradient elution (0~10min, 5%~15%A；10~
20min, 15%~25%A；20~30min, 25%~35%A)；
Flow velocity is 1.00ml/min；
Column temperature is 25 DEG C；
Record the characteristic spectrum of 16 batches of Dan peach kernel medicinal materials.The result is shown in Figure 1.
4) characteristic spectrum is analyzed
5 characteristic peaks are presented in Dan peach kernel medicinal material characteristic spectrum, wherein when 2 peaks retain with corresponding object of reference peak respectively
Between it is identical, peak corresponding with amarogentin object of reference be the peak S, peak corresponding with tryptophan object of reference be peak 2, calculate each characteristic peak
With the relative retention time at the peak S, relative retention time is within ± the 8% of specified value.Specified value are as follows: 0.880 (peak 1),
0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
2 Dan peach kernel medicine materical crude slice characteristic spectrum of embodiment
1) preparation of reference solution:
Amarogentin, tryptophan product are taken, respectively plus every 1ml amarogentin containing 1mg, 0.01mg tryptophan is made in methanol
Reference solution；
2) preparation of test solution:
16 crowdes of each 1g of Dan peach kernel medicine materical crude slice are taken respectively, and accurately weighed, accurate respectively that water 50ml is added, weighed weight boils 30
Minute, it lets cool, then weighed weight, the weight of less loss is supplied with water, is shaken up, filter, as test solution；
3) measurement of characteristic spectrum:
It is accurate respectively to draw reference solution and 10 μ l of test solution, liquid chromatograph is injected, chromatogram is obtained；
Chromatographic condition is as follows:
Detection wavelength: 214nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: methanol (A) and 0.1% phosphoric acid solution (B), gradient elution (0~10min, 5%~15%A；10~
20min, 15%~25%A；20~30min, 25%~35%A)；
Flow velocity is 1.00ml/min；
Column temperature is 25 DEG C；
Characteristic spectrum measurement is carried out to 16 batches of Dan peach kernel medicine materical crude slice, records map.As a result see Fig. 2.
4) characteristic spectrum is analyzed:
5 characteristic peaks are presented in Dan peach kernel medicine materical crude slice characteristic spectrum, wherein when 2 peaks retain with corresponding object of reference peak respectively
Between it is identical, peak corresponding with amarogentin object of reference be the peak S, peak corresponding with tryptophan object of reference be peak 2, calculate each characteristic peak
With the relative retention time at the peak S, relative retention time is within ± the 8% of specified value.Specified value are as follows: 0.880 (peak 1),
0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
3 Dan peach kernel standard decoction characteristic spectrum of embodiment
1) preparation of reference solution:
Amarogentin, tryptophan product are taken, respectively plus every 1ml amarogentin containing 1mg, 0.01mg tryptophan is made in methanol
Reference solution；
2) preparation of test solution:
16 crowdes of Dan peach kernel standard decoction each 0.2g are taken respectively, it is accurately weighed, it sets in stuffed conical flask, it is accurate respectively to be added
70% methanol 25ml, close plug, weighed weight, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes are let cool, then weighed heavy
Amount, the weight of less loss is supplied with 70% methanol, is shaken up, and is filtered, subsequent filtrate is taken, as test solution；
3) measurement of characteristic spectrum:
It is accurate respectively to draw reference solution and 10 μ l of test solution, liquid chromatograph is injected, chromatogram is obtained；
Chromatographic condition is as follows:
Detection wavelength: 214nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: methanol (A) and 0.1% phosphoric acid solution (B), gradient elution (0~10min, 5%~15%A；10~
20min, 15%~25%A；20~30min, 25%~35%A)；
Flow velocity is 1.00ml/min；
Column temperature is 25 DEG C；
4) characteristic spectrum is analyzed:
Characteristic spectrum measurement is carried out to 16 batches of Dan peach kernel standard decoctions, records map.As a result see Fig. 3.
5 characteristic peaks are presented in Dan peach kernel standard decoction characteristic spectrum, wherein 2 peaks are protected with corresponding object of reference peak respectively
Stay the time identical, peak corresponding with amarogentin object of reference is the peak S, and peak corresponding with tryptophan object of reference is peak 2, calculates each spy
The relative retention time at peak and the peak S is levied, relative retention time is within ± the 8% of specified value.Specified value are as follows: 0.880 (peak
1), 0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
4 Dan peach kernel granule characteristic spectrum of embodiment
1) preparation of reference solution:
Amarogentin, tryptophan product are taken, respectively plus every 1ml amarogentin containing 1mg, 0.01mg tryptophan is made in methanol
Reference solution；
2) preparation of test solution:
3 crowdes of granule each 0.2g are taken respectively, it is accurately weighed, it sets in stuffed conical flask, it is accurate respectively that 70% methanol is added
25ml, close plug, weighed weight, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes are let cool, then weighed weight, with 70%
Methanol supplies the weight of less loss, shakes up, and filtration takes subsequent filtrate, as test solution；
3) measurement of characteristic spectrum:
It is accurate respectively to draw reference solution and 10 μ l of test solution, liquid chromatograph is injected, chromatogram is obtained；
Chromatographic condition is as follows:
Detection wavelength: 214nm；
Chromatographic column: C18 chromatographic column；
Mobile phase: methanol (A) and 0.1% phosphoric acid solution (B), gradient elution (0~10min, 5%~15%A；10~
20min, 15%~25%A；20~30min, 25%~35%A)；
Flow velocity is 1.00ml/min；
Column temperature is 25 DEG C；
Characteristic spectrum measurement is carried out to 3 batches of granules, records map.As a result see Fig. 4.
4) characteristic spectrum is analyzed:
5 characteristic peaks are presented in Dan peach kernel granule characteristic spectrum, wherein 2 peaks are protected with corresponding object of reference peak respectively
Stay the time identical, peak corresponding with amarogentin object of reference is the peak S, and peak corresponding with tryptophan object of reference is peak 2, calculates each spy
The relative retention time at peak and the peak S is levied, relative retention time is within ± the 8% of specified value.Specified value are as follows: 0.880 (peak
1), 0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
5 Dan peach kernel medicinal material of embodiment-medicine materical crude slice-extract and granule compare map
Using similarity evaluation (2012 editions) respectively by 16 batches of Dan peach kernel medicines in embodiment 1
Material characteristic spectrum synthesizes a medicinal material and compares map, and 16 batches of Dan peach kernel medicine materical crude slice characteristic spectrums synthesize a medicine materical crude slice pair in embodiment 2
According to map, 16 batches of Dan peach kernel standard decoction characteristic spectrums synthesize a standard decoctions and compare map in embodiment 3,3 in embodiment 4
It criticizes Dan peach kernel granule characteristic spectrum and synthesizes a granule control map.By the above peach kernel medicinal material, medicine materical crude slice, standard soup
Agent, Dan peach kernel granule control map compare, and as a result see Fig. 5.
The result shows that: 5 characteristic peaks in medicine materical crude slice and standard decoction, Dan peach kernel granule can detect in medicinal material,
Illustrate that these ingredients all derive from medicinal material, while knowing Dan peach kernel medicinal material, Dan peach kernel medicine materical crude slice, Dan peach kernel standard soup from control map
Agent, the chemical component of Dan peach kernel granule are identical, and material base is consistent.
Beneficial effects of the present invention are illustrated below by way of test example:
1 characteristic spectrum methodology of test example
1, chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Methanol is mobile phase A；Using 0.1% phosphoric acid solution as Mobile phase B, the regulation according to the form below carries out gradient elution；Flow velocity is every point
Clock 1.0ml；Column temperature is 25 DEG C；Detection wavelength is 214nm.Number of theoretical plate is calculated by amarogentin peak should be not less than 3000.Such as table 1
It is shown:
The eluent gradient that table 1 is drafted
2, the preparation of reference solution
Take amarogentin, tryptophan product appropriate, respectively plus every 1ml 1mg containing amarogentin, tryptophan is made in methanol
The reference substance solution of 0.01mg to get.
3, the preparation of test solution
Dan peach kernel granule 0.2g is taken, it is accurately weighed, it sets in stuffed conical flask, 70% methanol 25ml is added in precision, close
Plug, weighed weight, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes let cool, then weighed weight, are supplied with 70% methanol
The weight of less loss, shakes up, filtration, take subsequent filtrate to get；
Peach kernel control medicinal material 1g is taken, it is accurately weighed, it is separately added into water 50ml, weighed weight is boiled 30 minutes, is let cool, then
Weighed weight is supplied the weight of less loss with water, is shaken up, filtration, as control medicinal material solution.
4, measuring method
Precision draw 10 μ l of test solution, inject liquid chromatograph, measurement to get.
5, methodological study
5.1 specificities are investigated
Test solution and negative control solution are prepared by the method for drafting, is detected.As a result see Fig. 6.
5.2 precision test
Same test solution (lot number: SY1804001) is taken, by chromatographic process continuous sample introduction 6 times is determined, 10 μ l every time,
The retention time and peak area result for calculating each chromatographic peak are shown in Table 2 and table 3 respectively.
2 precision of table-characteristic peak retention time
3 precision of table-characteristic peak peak area
The result shows that the RSD of each characteristic peak retention time is 0.03%~0.04%, the RSD of each characteristic peak peak area exists
0.11%~1.50%, the instrument precision is good.
5.3 repeatability are investigated
Precision weighs 6 parts of peach kernel granule (lot number: SY1804001), is prepared and is measured by experimental method is drafted.
It is shown in Table 4,5.
Repeated investigation-characteristic peak relative retention time the ratio of table 4
Repeated investigation-characteristic peak relative peak area the ratio of table 5
The result shows that the RSD of each characteristic peak relative retention time is 0.01%~0.18%, each characteristic peak relative peak area
RSD 0.48%~1.02%.
5.4 Intermediate precisions are investigated
5.4.1 different instruments are investigated
On the basis of the experiment condition drafted above, precision weighs Dan peach kernel granule (lot number: SY1804001) respectively
Three parts, test solution is prepared, is surveyed on Waters, Shimadzu 20AT, 1260 type high performance liquid chromatograph of Agilent respectively
Fixed (chromatographic column is (2) 250 × 4.6mm of Agilent 5HC-C18).See Fig. 7~9, table 6,7.
The different instrument investigation-characteristic peak relative retention time ratios of table 6
The different instrument investigation-characteristic peak relative peak area ratios of table 7
The result shows that the RSD of each characteristic peak relative retention time exists when being detected with above-mentioned 3 kinds of instruments to test sample
0.51%~1.82%；The RSD of each characteristic peak relative peak area is 8.01%~14.89%.
5.4.2 different personnel and time are investigated
On the basis of the experiment condition drafted above, by different personnel (A, B), at different time (T1, T2), precision claims respectively
Each two parts of Dan peach kernel granule (lot number: SY1804001) is taken, test sample is prepared, is measured.It is shown in Table 8,9.
The different personnel of table 8 and time investigation-characteristic peak relative retention time ratio
The different personnel of table 9 and time investigation-characteristic peak relative peak area ratio
The result shows that under different sample preparation personnel and different sample preparation time conditions, when each characteristic peak retains relatively
Between RSD 0.01%~0.12%；The RSD of each characteristic peak relative peak area is 0.36%~1.15%.
5.5 durabilities are investigated
5.5.1 chromatographic column durability is investigated
It is respectively 5 μm of 4.6 × 250mm of Agilent HC-C18 to chromatographic column on the basis of the experiment condition drafted above
(Shimadzu), phenomenex Lna 5 μm of C18 (2) 250 × 4.6mm of 100A 5micron (Féraud door), Diamonsil C18
(2) 5 μm of 250 × 4.6mm (enlightening horse) are investigated.See Figure 10, table 10,11.
10 chromatographic column durability investigation of table-characteristic peak relative retention time
11 chromatographic column durability investigation of table-characteristic peak relative peak area ratio
The result shows that being detected with above-mentioned 3 kinds of chromatographic columns to sample, the RSD of characteristic peak relative retention time exists
4.88%~5.52%, the RSD of characteristic peak relative peak area is 4.52%~18.25%.
5.5.2 study on the stability
On the basis of the experiment condition drafted above, take same test solution, respectively at 0h, 4h, 8h, 12h, 18h,
It is measured when for 24 hours.It is shown in Table 12,13.
12 24 hours study on the stability of table-characteristic peak retention time
13 24 hours study on the stability of table-characteristic peak peak area ratio
The result shows that the RSD of corresponding characteristic peak retention time is 0.10%~1.13%, characteristic peak peak area
RSD is 0.41%~2.38%.
In conclusion the RSD of each characteristic peak relative retention time meets the requirements in above every investigation, this method is good
It is good.
The determination of 5.6 characteristic peaks and the foundation for compareing map
The measurement for carrying out characteristic spectrum to Dan peach kernel granule using the method drafted, calculates relative retention time, phase
To peak area and opposite peak height.See Figure 11, table 14~16.
14 Dan peach kernel granule relative retention time of table
15 Dan peach kernel granule relative peak area of table
16 Dan peach kernel granule of table is with respect to peak height
According to the principle that relative retention time is stable and each batch sample can detect and peak is relatively high, 5 have been selected altogether
A preferable peak of repeatability is as characteristic peak.The result shows that when peak 3 is as the peak S, 3 batch Dan peach kernel granule characteristic peaks
Relative peak area RSD 1.07%~4.11%, 3 batch Dan peach kernel granule characteristic peak with respect to peak height RSD 1.38%~
4.27%, 35 characteristic peak relative retention time RSD of batch Dan peach kernel granule are respectively less than 2.0%.
The formulation of 5.7 relative retention time specified value limits
Methodology respectively investigates project and verification result summarizes and sees Figure 12, table 17~18:
17 methodology projects result RSD% of table summarizes standard-relative retention time
18 methodology projects result RSD% of table summarizes standard-relative peak area
Different chromatographic column relative retention times are affected as seen from the above table, and RSD% is respectively less than 6%, for the side of increase
The reproducibility and applicability of method, therefore it is 8% that Jiang Gefeng and the relative retention time specified value at peak 3, which are fixed tentatively,.
Final regulation: should be presented 5 characteristic peaks in test sample characteristic spectrum, wherein 2 peaks should respectively with corresponding reference
Object peak retention time is identical, and peak corresponding with amarogentin object of reference is the peak S, and peak corresponding with tryptophan object of reference is peak 2, meter
The relative retention time of each characteristic peak Yu the peak S is calculated, relative retention time should be within ± the 8% of specified value.Specified value are as follows:
0.880 (peak 1), 0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113 (peaks 4), 1.144 (peaks 5).
Pass through characteristic spectrum methodological study, it is thus identified that the detection method can be used for Dan peach kernel medicinal material-medicine materical crude slice-extract and
The quality testing of granule, can accurate response Dan peach kernel medicinal material-medicine materical crude slice-extract and the multicomponent integral face of granule
Looks.
Claims (10)
1. a kind of characteristic spectrum detection method of Dan peach kernel preparation, it is characterised in that: it is detected using high performance liquid chromatography,
Its operating procedure is as follows:
1) preparation of reference solution: taking amarogentin, tryptophan product, and respectively plus methanol dissolves, as reference solution；
2) preparation of test solution:
A medicinal material or medicine materical crude slice test sample: taking test sample, extracting in water, and filtering takes filtrate to get test solution；
B standard decoction or granule test sample: taking test sample, and 70% methanol is added to extract, and filtering takes filtrate, both obtains test sample
Solution；
3) reference solution being drawn respectively and test solution injecting liquid chromatograph, chromatographic condition is as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Mobile phase: using methanol as mobile phase A, with 0.1% phosphoric acid
Solution is Mobile phase B gradient elution；Gradient elution program is as follows:
4) characteristic spectrum is analyzed.
2. quality determining method according to claim 1, it is characterised in that: step 1) the reference substance amarogentin, color
Propylhomoserin concentration is respectively solution of every 1ml containing 1mg, 0.01mg.
3. quality determining method according to claim 1, it is characterised in that: the matter of medicinal material described in step a or medicine materical crude slice and water
Amount volume ratio is 1g:50mL.
4. quality determining method according to claim 1, it is characterised in that: be extracted as decocting described in step a and extract, decoct
Boiling time is 30min.
5. quality determining method according to claim 1, it is characterised in that: standard decoction described in step b or granule
Mass volume ratio with 70% methanol is 1g:125mL.
6. quality determining method according to claim 1, it is characterised in that: be extracted as ultrasonic extraction described in step b, ultrasound
Extract power 600W, frequency 40kHz, time 30min.
7. quality determining method according to claim 1, it is characterised in that: be injected separately into liquid chromatogram described in step 3)
The reference solution and test solution amount of instrument are 10~20 μ l, preferably 10 μ l.
8. quality determining method according to claim 1, it is characterised in that: the wavelength of the step 3) chromatographic condition is
214nm, column temperature are 25 DEG C, flow rate of mobile phase 1.00ml/min, and number of theoretical plate is calculated by amarogentin peak should be not less than 3000.
9. quality determining method according to claim 1, it is characterised in that: step 3) the octadecylsilane bonded silica
Glue chromatographic column be 5 μm of 5 μm of 4.6 × 250mm of Agilent HC-C18, phenomenex Lna C18 (2) 100A 250 ×
(2) 5 μm of 5 micron of 4.6mm, Diamonsil C18 250 × 4.6mm, preferably 5 μm 4.6 of Agilent HC-C18 ×
250mm。
10. detection method according to claim 1, it is characterised in that: 5 should be presented in the characteristic spectrum of the step 4)
Characteristic peak, wherein 2 peaks should be identical as corresponding object of reference peak retention time respectively, peak corresponding with amarogentin object of reference is
The peak S, peak corresponding with tryptophan object of reference are peak 2, calculate the relative retention time of each characteristic peak Yu the peak S, when retaining relatively
Between should be within ± the 8% of specified value.Specified value are as follows: 0.880 (peak 1), 0.901 (peak 2), 1.000 (peak 3, the peaks S), 1.113
(peak 4), 1.144 (peaks 5).
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