CN102680626A

CN102680626A - Quantitative detection method for characteristic components in Shenmai injection

Info

Publication number: CN102680626A
Application number: CN2012101518466A
Authority: CN
Inventors: 凌益平; 王颖; 凌伫怡; 柴建国; 王木兰; 陈莉
Original assignee: ZHENGDA QINGCHUNBAO PHARMACEUTICAL CO Ltd
Current assignee: ZHENGDA QINGCHUNBAO PHARMACEUTICAL CO Ltd
Priority date: 2012-05-09
Filing date: 2012-05-09
Publication date: 2012-09-19

Abstract

The invention discloses a liquid chromatography mass spectrometry quantitative detection method for determining effective components in Shenmai injection. A sample disclosed by the invention dispenses with a complex pretreatment process and is obtained by filtering through a microfiltration membrane with 0.2mum; in liquid chromatography separation, a water-acetonitrile system is used as a mobile phase and gradient elution is carried out; and the detection is realized by using a mass detector; and quantitative detection is carried out at the peak area by adopting an external reference method. The quantitative detection method disclosed by the invention has the advantages of high sensitivity, favorable accuracy and precision and simplicity, convenience and quickness in operation; and the real-time detection in the production process of medicines is better realized.

Description

The quantitative detecting method of characteristic chemical constituent in a kind of Shenmai injection

Technical field

The present invention relates to the quantitative detecting method of characteristic chemical constituent in a kind of traditional Chinese medicine injection, specifically in the Shenmai injection tuber of dwarf lilyturf medicinal material characteristic chemical constituent borneol glycosides method for quantitatively determining.

Background technology

Shenmai injection effective constituent is mainly saponin(e (be about solid content 7.5%), flavonoids and saccharide compound; Wherein saponins has been found to be 39 kinds of ginsenoside constituents of representative and to be 7 kinds of ophiopogonin constituents of representative with the ophiopogonin D with panax saponin-Re, Rb1, Rb2, Rg1, Rg3 etc. at present, and flavonoids has found it then mainly is that what to contain in the tuber of dwarf lilyturf is 8 kinds of homoisoflavone constituents of representative with methyl dihydro tuber of dwarf lilyturf homoisoflavone B, 2 beta-hydroxy Methylophiopogonanone A Bs, 2 Alpha-hydroxy Methylophiopogonanone A Bs etc. at present.

Existing operative norm is the national drug standards (revision) promulgation part WS3-B-3428-98-2004; Effective constituent sanchinoside E1, the Rg1 of pair red ginseng (or red ginseng had with the tuber of dwarf lilyturf), the discriminating of Re reference substance during differentiating, it are only arranged; Mainly concentrate on the contained effective constituent of red ginseng for the discriminating of Shenmai injection and the research of active constituent content measuring method in the early time, less to the effective constituent research tuber of dwarf lilyturf.

Chinese patent CN101745024A discloses a kind of borneol glycosides that detects in the medicinal material tuber of dwarf lilyturf that utilizes first and has differentiated the technical scheme in the Shenmai injection, discloses the high performance liquid chromatogram quantitative detecting method of borneol glycosides simultaneously.Shenmai injection has been filled up in this invention does not have differentiate the blank of item the tuber of dwarf lilyturf for a long time, makes the Quality Control level of Shenmai injection bring up to a new step.

In order further to improve the quality control level of Shenmai injection; The detection of borneol glycosides is applied to the overall process of this production; The applicant invented a kind of can be in short-term, need not to obtain the method for borneol glycosides content in the soup under the condition that complex sample handles.

Summary of the invention

The purpose of this invention is to provide a kind ofly in short-term, need not to obtain the method for borneol glycosides content in the soup under the condition that complex sample handles.

Technical scheme of the present invention is for utilizing LC-MS technology, and through simple sample preparation, liquid phase separation, the Mass Spectrometer Method process is monitored the content of borneol glycosides in intermedium and the finished product in the Shenmai injection production procedure in real time.

(1) sample preparation: directly extract intermedium or finished product in the production procedure, behind 0.2 μ m filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

(2) liquid phase separation

Liquid phase chromatogram condition:

1) liquid chromatograph: Agilent 1290UHPLC

2) chromatographic column: PoroShell EC-C18,2.7 μ m, 2.1 * 100mm

3) column temperature is 40 ℃

4) moving phase: A water; The B acetonitrile

5) flow velocity: 0.3mL/min

6) sampling volume: 1 μ L

7) gradient elution program:

Table 1 eluent gradient program list

(3) Mass Spectrometer Method

The mass spectrum condition:

1) level Four bar flight time mass spectrum system: Agilent 6520QTOF MS;

2) ion gun: electric spray ion source (ESI), negative ion monitoring (ESI-);

3) spraying source gas flow rate: 10L/min, the temperature of desolvating is 325 ℃;

4) atomization gas pressure: 40psi;

5) WV: 180V;

Select ion condition: m/z447 [M-H] ^-, m/z483 [M-H] ^-And m/z493 [M-H] ^-The ESI signal of three adduct ions adds up.Retention time is seen Fig. 1, and Fig. 2 is seen in the stack of adduct ion peak area.

Density calculating method: with peak area or peak height method, calculate borneol glycosides content in the Shenmai injection by internal standard method

Calibration curve preparation and mensuration: it is an amount of to get borneol glycosides reference substance.Being dissolved in water is diluted to 0.5mg/mL, as storing solution; Be diluted to 1 μ g/mL with water successively, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard sequences are got 1 μ L respectively and are measured, each concentration point replication 3 times, the result sees Fig. 3;

Sensitivity determination: get the 1 μ L sample introduction analysis of 1 μ g/mL borneol glycosides reference substance solution, the EICs of 3 quota ions calculates signal to noise ratio (S/N ratio) and is respectively S/N 422 (m/z447), S/N 175 (m/z483), S/N 2034 (m/z493).

Useful result of the present invention is: the inventive method is highly sensitive, accuracy and precision are good, and specificity and authenticity are high, and very easy and can be easy to actual detected and use, and the extraordinary real-time monitoring of satisfying in the drug production process.The method that the method for employing LC-MS is monitored the content of the borneol glycosides in the Shenmai injection is not in real time seen corresponding report at present as yet.

Description of drawings

Accompanying drawing 1 is retention time figure

Accompanying drawing 2 is adduct ion peak area stacking diagrams

Accompanying drawing 3 is correction graph

The practical implementation method

The specific embodiments of invention is in order to be illustrated more clearly in the present invention, is not the restriction to technical characterictic of the present invention.

Embodiment:

Sample source: produced by Zhengda Qingchunbao Pharmaceutical Co., Ltd, its production technology is all carried out according to the current national drug standards, and six parts of soups are respectively: finished product, lot number: 1103058; Do not add the semi-manufacture of isotonic regulator, lot number: 1101178; The tuber of dwarf lilyturf herbal extract, lot number: 11062; Red ginseng alcohol extract tuber of dwarf lilyturf amalgamation liquid, lot number: 11063; Soup after the extract charcoal absorption, lot number: 11064; The parenteral solution dilution, lot number: 11065

Reagent: HPLC analyzes and uses acetonitrile to be Merck reagent Company products; Water is water for injection;

Borneol glycosides reference substance is oneself to separate the preparation gained, and medicine inspecting institute demarcates through Zhejiang Province, and purity is 99.7%;

Sample pretreatment: get six parts of soups in the Shenmai injection production run, behind 0.2 μ m filtering with microporous membrane, it is subsequent use to get subsequent filtrate;

Liquid phase separation:

Liquid chromatograph: Agilent 1290UHPLC comprises binary UHV (ultra-high voltage) gradient pump, automatic sampler, column oven; Chromatographic column: PoroShell EC-C18,2.7 μ m, 2.1 * 100mm; Column temperature: 40 ℃; Moving phase: A water: B acetonitrile; Flow velocity: 0.3mL/min; Sampling volume: 1 μ L.

Gradient elution program (in percent by volume): 0-0.5min, 5%B; 0.5-15min, 5-30%B; 15-20min, 30-80%B; 20-25min, 80%B;

Mass Spectrometer Method: level Four bar flight time mass spectrum system: Agilent 6520QTOF MS; Ion gun: electric spray ion source (ESI), negative ion monitoring (ESI-); Spraying source gas flow rate: 10L/min, vaporizer temperature: 325 ℃; Atomization gas pressure: 40psi; WV: 180V.

Linear relationship is investigated and the calibration curve preparation

The standard items storing solution: it is an amount of to get borneol glycosides reference substance, and being dissolved in water is diluted to 0.5mg/mL.Be diluted to 1 μ g/mL with water successively, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard sequences, altogether 5 concentration reference substance solution.The above standard solution of accurate respectively absorption 1 μ l is measured under above-mentioned chromatographic condition, is ordinate Y with the peak area, is that horizontal ordinate X carries out linear regression with borneol glycosides content, and the result sees accompanying drawing 3.Getting linear equation is:

lnY＝16.522572+0.848575*ln(X)-0.055856*ln(X)^2。

Precision is investigated

Replica test is got each 3 parts of same batch of soups (lot number 1101178 and 1103058); Make each 3 parts of need testing solution according to preprocess method, the accurate respectively 1 μ l that draws measures; Calculate content (μ g/ml); Test result is seen table 3, and RSD (n=3) is respectively 0.98% and 0.72%, shows that this method repeatability is good.

Table 2 repeated experiment data list (μ g/ml)

Middle precision test is got each 6 parts of same batch these article (lot number 1103058) by two analysis personnel respectively in same date not; Make 12 parts of need testing solutions altogether according to preprocess method; Measure according to set detection method in same date not, calculate content (μ g/ml), test result is seen table 3; RSD (n=12) is 1.50%, shows that the middle precision of this method is good.

Precision experimental data tabulation (μ g/ml) in the middle of the table 3

Stability test

Get need testing solution that same batch these article (lot number 1103058) make as requested respectively 0,1.5,3,4.5,6,7.5,9,10.5, the accurate 1 μ l of absorption of 12h measures; Calculate content (μ g/ml); Test result is seen table 4; RSD (n=9) is 2.60%, shows that test liquid is stable in 12h at least.

Table 4 stability experiment data list (μ g/ml)

Mean value: 3.51 μ g/ml; RSD=2.60%

Durability (investigating the influence of column temperature)

Getting same batch of need testing solution (lot number 1103058) is 30 ℃ during with 50 ℃ through changing column temperature, measures in accordance with the law, distinguishes sample introduction 2 times; Each accurate respectively 1 μ l that draws, the peak area of mensuration borneol glycosides calculates content (μ g/ml); Test result is seen table 5, and RSD (n=6) is respectively 1.68%.Show that it all is accurately that the mensuration column temperature is set in 30～50 ℃ of results.

Table 5. durability is investigated: the influence that column temperature is measured content (μ g/ml)

Sample determination

Get the need testing solution that the soup of each batch makes according to the pre-service preparation method, measure, in accordance with the law according to borneol glycosides content in the calibration curve calculation sample.The result sees table 6.

The content of borneol glycosides in table 6. Shenmai injection (μ g/ml)

Claims

1. the determination method by liquid chromatography-electrospray mass of borneol glycosides in the Shenmai injection is characterized in that through sample preparation, liquid phase separation, three steps of Mass Spectrometer Method:

(1) sample preparation: directly extract intermedium or finished product in the production procedure, behind 0.2 μ m filtering with microporous membrane, get subsequent filtrate and change sample introduction bottle sample introduction over to;

(2) liquid phase separation:

Liquid-phase condition is:

1) liquid chromatograph: Agilent 1290 UHPLC

2) chromatographic column: PoroShell EC-C18,2.7 μ m, 2.1 * 100mm

3) column temperature is 40 ℃

4) moving phase: A water; The B acetonitrile

5) flow velocity: 0.3mL/min

6) sampling volume: 1 μ L

7) gradient elution program (in percent by volume): 0-0.5min, 5%B; 0.5-15min, 5-30%B; 15-20mi n, 30-80%B; 20-25mi n, 80%B;

(3) Mass Spectrometer Method:

The mass spectrum condition is:

1) level Four bar flight time mass spectrum system: Agilent 6520QTOF MS;

2) ion gun: electric spray ion source (ESI), negative ion monitoring (ESI-);

4) atomization gas pressure: 40psi;

5) WV: 180V;

Select ion condition: m/z447 [M-H] ^-, m/z483 [M-H] ^-And m/z493 [M-H] ^-The ESI signal of three adduct ions adds up.

Density calculating method: with peak area or peak height method, calculate borneol glycosides content in the Shenmai injection by external standard method.