CN103760245B - HPLC-MS multi-index rapid detection method for illegally added cough and asthma relieving chemical components - Google Patents
HPLC-MS multi-index rapid detection method for illegally added cough and asthma relieving chemical components Download PDFInfo
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Abstract
The invention relates to an HPLC-MS (high performance liquid chromatography-mass spectrometry) multi-index rapid detection method for illegally added cough and asthma relieving chemical components. The key point of the method lies in that the control sample is a mixture of theophylline, sulfamethoxazole, chlorphenamine, diphenhydramine, Pentoxyverine, benproperine, prednisone acetate and diazepam. The invention establishes an efficient detection method for illegally added cough and asthma relieving chemical components. The method has no significant difference from single index detection item, detection limit and specificity, and has very high detection efficiency.
Description
Technical field
The present invention relates to the method for quick of illegal adding ingredient in a kind of health food and medicine, relate to the liquid-matter multiple index quick detecting method of the relieving cough and asthma class chemical composition of a kind of illegal interpolation in particular.
Technical background
LC-MS-MS (HPLC-MS) i.e. LC-MS technology.The piece-rate system of this technology is liquid chromatography, detection system is then mass spectrum, namely utilize the isolation technics of chromatogram, biased sample is separated into single material, after entering mass spectrum in order, these materials are broken into pieces by mass spectrographic mass analyzer, carried out the structure of amalyzing substances by mass detector, utilize the fracture mode judging key, by karyoplasmic ratio according to contrasting picture library accordingly, judge the structure of fragment ion, thus qualitative, quantitative is carried out to unknown sample.This LC-MS technology has embodied chromatogram and mass spectrographic mutual supplement with each other's advantages, chromatogram and mass spectrographic advantages are got up, allow people can utilize the features such as their high separating force, high sensitivity and high selectivity in analysis, the system of adding can provide contrast spectrogram, makes analytical test can more and more easier circle.LC-MS is the technology of comparative maturity, in current many fields as being widely used in pesticide residue analysis, food analysis, environmental analysis and Pharmaceutical Analysis etc.LC-MS system conventional on present analysis is divided into mass analyzer to classify and ion gun classification two large classes substantially from mass spectrum direction.Mass analyzer classification comprises level Four bar, flight time (TOF), Fourier transform mass spectrum: ion gun classification comprises atmosphere pressure chemical ion source (APCI), atmospheric pressure photoionization source (APPI), electric spray ion source (ESI), substance assistant laser desorpted ionized source (MALDI).
With the detecting device of Traditional liquid phase chromatogram, as UV or PAD compares, mass spectrum has the features such as highly sensitive and qualification function is strong, and add the analyst coverage of liquid chromatography comparatively extensively, LC-MS technology has become one of the most strong instrument in big event Chinese traditional medicine analysis aspect in recent years.Especially in recent years for the research of various medicine especially forbidden drug and metabolic product thereof, a large amount of bibliographical informations was had both at home and abroad.
Summary of the invention
But due to for a certain medicine or the addible similar chemical composition of health products very many, as detected one by one for each composition, not only testing cost is huge, needs a large amount of detection times simultaneously, is unfavorable for increasing work efficiency; Therefore the invention provides the liquid-matter multiple index quick detecting method of the relieving cough and asthma class chemical composition of a kind of illegal interpolation.Concrete operation steps is:
1) get the Chinese patent drug being equivalent to an oral dose or health food (solid pharmaceutical preparation needs porphyrize, liquid preparation direct sample), add methyl alcohol 25ml, ultrasonic process 10 minutes, filter with miillpore filter (0.45um), filtrate is suitably diluted with methyl alcohol, as need testing solution.
2) get theophylline, Sulfamethoxazole, chlorphenamine, diphenhydramine, pentoxyverine, benproperine, prednisone acetate, diazepam reference substance in right amount, add methyl alcohol respectively and make the solution of every 1ml containing 0.1mg, product solution in contrast.
3) upper machine testing: examination and qualification are carried out to sample by high performance liquid chromatography-flight time mass spectrum method for combined use.
High performance liquid chromatograph: waters AQUITY UPLC instrument;
Be that (chromatographic column is WATERS BEH C to filling agent with octadecylsilane chemically bonded silica
18, 100mm × 2.1mm, 1.7 μm);
With the methyl alcohol containing 0.1% formic acid for mobile phase A, with the ammonium formate solution of the 10mmol/L containing 0.1% formic acid for Mobile phase B, according to the form below 1 is to carry out gradient elution;
Table 1HPLC gradient elution mobile phase
Flow velocity: 0.3ml/min;
Column temperature: 40 DEG C; ;
Sample temperature: 15 DEG C;
Sample size: 1ul;
Time of-flight mass spectrometer: waters Q-TOF instrument;
Electron spray ionisation component (ESI);
The MS detection parameters: ESI+ scans;
Dry gas temperature: 350 DEG C;
Taper hole voltage: 35v;
Scan mode: first mass spectrometric full scan, second order ms full scan, sweep limit: 100 ~ 1000m/z;
4) identify:
In test sample chromatogram, occur and the identical chromatographic peak of each reference substance chromatographic retention in mixing reference substance, and the first mass spectrometric of this chromatographic peak LC-MS of test sample and second order ms all consistent with reference substance, namely judge that this test sample is as the positive.
In test sample chromatogram, there is not the chromatographic peak identical with each reference substance chromatographic retention in mixing reference substance, namely judge that this test sample is as negative.
Advantageous Effects of the present invention is: the present invention establishes a kind of relieving cough and asthma class chemical composition of illegal interpolation, efficient detection method, with single Indexs measure project, detectability and specificity are all without significant difference; And there is very high detection efficiency.
Accompanying drawing explanation
Fig. 1 reference substance extracts ion current chromatogram;
Fig. 2 reference substance first mass spectrometric figure;
Fig. 3 reference substance second order ms figure;
Fig. 4 negative sample (not detecting chlorphenamine) total ion current figure;
Fig. 5 positive (detecting chlorphenamine) total ion current figure;
Fig. 6 positive (having detected chlorphenamine) extracts ion flow graph (275.132,0.02Da);
Fig. 7 positive (detecting chlorphenamine) first mass spectrometric figure;
Fig. 8 positive (detecting chlorphenamine) second order ms figure;
Wherein, described in Fig. 1-3, the chemical composition of the correspondence of numbering is: 1-theophylline, 2-Sulfamethoxazole, 3-chlorphenamine, 4-diphenhydramine, and 5-sprays Trotskyite woods, 6-benproperine, 7-prednisone acetate, 8-diazepam.
Embodiment
The investigation of embodiment 1 chromatographic condition
1) selection of test sample extraction conditions
Investigated the positive that methyl alcohol and acetonitrile two kinds of solvent extraction parts are illegally added respectively, result shows, methyl alcohol and acetonitrile all can extract target component, but due to the cheap and less pollution of methyl alcohol, therefore adopt the method for the ultrasonic extraction of methyl alcohol.Investigated Strata-X, Strata-X-W, HLB and PCX tetra-kinds of solid phase extraction columns extract sample purification effect to methyl alcohol respectively, result all has stronger adsorption effect to target component, causes the recovery low, therefore does not adopt the step of Solid-Phase Extraction.
2) selection of mobile phase
Adopt the chromatographic condition in summary of the invention, aqueous phase has investigated formic acid respectively, formic acid-triethyl amine, formic acid-ammonium formate, formic acid ammonium formate-triethylamine system, result only adopts formate ion efficiency lower, the system peak type adding triethylamine is better, but ion depression effect is obvious, and after adding ammonium formate, Ionization Efficiency obviously strengthens, and peak type is better.Therefore adopt formic acid-ammonium formate system.Organic phase compares methyl alcohol, acetonitrile, different proportion methanol-acetonitrile mixed solution respectively, and result adopts methanol-acetonitrile mixed solution peak type symmetrical, sharp-pointed.Repeatedly gradient is adjusted, finally obtain good separating effect.(see figure 1)
3) investigation of different column chromatography separation case
Investigated separation case (1) the Agilent ZORBAX RRHD Eclipse Plus C of chromatographic column to composition to be measured of three different brands simultaneously
18post (100mm × 3.0mm, 1.8 μm), (2) Kromasil C
18(100mm × 2.1mm, 1.8 μm), (3) Waters ACQUITY HSS T3C
18post (100mm × 2.1mm, 1.8 μm), (4) Waters ACQUITY BEH C
18post (100mm × 2.1mm, 1.7 μm) test card bright target component chromatographic peak peak shape is better.
4) specificity is investigated
Carried out specificity investigation to ingredient in some positive sample, use the corresponding formulation and kind that do not contain additive as negative control, result shows, positive can detect the identical chromatographic peak of retention time, and negative sample is all without corresponding chromatographic peak; Specifically as shown in Figure 2.
5) detectability test
Get reference substance in above-mentioned 11 appropriate, add the reference substance solution injection liquid chromatography that methyl alcohol is mixed with variable concentrations, measure in accordance with the law, by 3 times of snr computation detectabilities, the lowest detectable limit of 11 kinds of reference substances is all lower than 0.01 μ gmL
-1.
6) mass spectral characteristic is determined
The first mass spectrometric molecular ion peak of reference substance and part illegal interpolation sample thereof and the main fragmention of second order ms are as shown in Table 2 and Figure 3.
Table 28 kind of the illegal mass ions peak added
The detection of embodiment 2 sample
Get certain health food commercially available being equivalent to an oral dose, porphyrize, liquid preparation direct sample, after adding the ultrasonic extraction of methyl alcohol, put less than 10 DEG C temperature placements and spend the night, after clarifying, get supernatant miillpore filter (0.22um) to filter, suitably dilute, as need testing solution.
Adopt the LC-MS described in embodiment preferred, by high performance liquid chromatography-flight time mass spectrum method for combined use, examination and qualification are carried out to sample.Find this sample to contain and illegally add chlorphenamine.Specifically as shown in Fig. 4,5,6,7,8.
Claims (2)
1. the illegal liquid-matter multiple index quick detecting method adding relieving cough and asthma class chemical composition, comprises the following steps:
1) sample is prepared;
2) by high performance liquid chromatography-flight time mass spectrum method for combined use, examination and qualification are carried out to sample;
3) high-efficient liquid phase chromatogram of the high-efficient liquid phase chromatogram of sample and a second order ms figure and reference substance and a second order ms figure are compared, identify and illegally add relieving cough and asthma class chemical composition;
It is characterized in that: described reference substance is the potpourri of theophylline, Sulfamethoxazole, chlorphenamine, diphenhydramine, pentoxyverine, benproperine, prednisone acetate and diazepam;
Wherein, step 2) described in chromatographic condition be:
High performance liquid chromatograph: waters AQUITY UPLC instrument;
Chromatographic column: take octadecylsilane chemically bonded silica as filling agent, 100 mm × 2.1 mm, 1.7 μm;
With the methyl alcohol containing 0.1% formic acid for mobile phase A, with the ammonium formate solution of the 10mmol/L containing 0.1% formic acid for Mobile phase B, according to the form below is to carry out gradient elution;
During 0-6min, the percent by volume of mobile phase A is changed to 30% → 80%, and Mobile phase B percent by volume is changed to 70% → 20%;
During 6-7min, the percent by volume of mobile phase A is 80%, and Mobile phase B percent by volume is 20%;
During 7-8min, the percent by volume of mobile phase A is changed to 80% → 30%, and Mobile phase B percent by volume is changed to 20% → 70%;
During 8-10min, the percent by volume of mobile phase A is 30%, and Mobile phase B percent by volume is 70%;
Flow velocity: 0.3ml/min;
Column temperature: 40 DEG C;
Sample temperature: 15 DEG C;
Sample size: 1ul;
Time of-flight mass spectrometer: waters Q-TOF instrument;
Electron spray ionisation component (ESI);
The MS detection parameters: ESI
+scanning;
Dry gas temperature: 350 DEG C;
Taper hole voltage: 35v;
Scan mode: first mass spectrometric full scan, second order ms full scan, sweep limit: 100 ~ 1000 m/z.
2. liquid-matter the multiple index quick detecting method of the relieving cough and asthma class chemical composition of illegal interpolation according to claim 1, is characterized in that:
The preparation method of the sample described in step 1) is: get the Chinese patent drug or health food that are equivalent to an oral dose, add methyl alcohol 25ml, ultrasonic process 10 minutes, and filter with 0.45 μm of miillpore filter, filtrate is suitably diluted with methyl alcohol, as need testing solution; Wherein, solid pharmaceutical preparation needs porphyrize, liquid preparation direct sample.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104931601B (en) * | 2015-05-12 | 2017-02-08 | 广西壮族自治区梧州食品药品检验所 | Method for simultaneously detecting multiple chemical medicines illegally added into liquid health-care food |
| CN104833757B (en) * | 2015-05-12 | 2017-02-08 | 广西壮族自治区梧州食品药品检验所 | Method of simultaneously determining multiple chemical drugs illegally added in soft capsule health food |
| CN105158358B (en) * | 2015-08-18 | 2017-09-29 | 江苏出入境检验检疫局动植物与食品检测中心 | The method for detecting the 42 kinds of chemicals illegally added in Chinese patent drug and health products simultaneously |
| CN107677743A (en) * | 2017-09-28 | 2018-02-09 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A kind of method of chlorphenamine maleate and other chemicalses in detection herbal beverage |
| CN107884503A (en) * | 2017-11-01 | 2018-04-06 | 广西壮族自治区食品药品检验所 | 22 kinds of detection methods for illegally adding relieving cough and asthma class medicine in asthma-relieving bolus of gecko |
| CN108051534B (en) * | 2017-11-20 | 2019-10-18 | 中山大学 | A kind of method of the 132 kinds of chemicals illegally added in rapid screening Chinese patent drug and health care product |
| CN107741465B (en) * | 2017-11-27 | 2020-12-04 | 浙江公正检验中心有限公司 | Method for detecting residual quantity of 18 illegally added hypoglycemic and antihypertensive drugs in health care product |
| CN113791162B (en) * | 2021-11-16 | 2022-02-08 | 潍坊市检验检测中心 | Method for determining 35 chemical components illegally added in patch for relieving cough and asthma of children |
| CN117214125B (en) * | 2023-11-09 | 2024-01-26 | 南京盛略科技有限公司 | Liquid component detection system and method based on detection optical fiber |
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