CN113791162B - Method for determining 35 chemical components illegally added in patch for relieving cough and asthma of children - Google Patents

Abstract

The invention provides a method for determining 35 chemical components illegally added in a patch for relieving cough and asthma of children, which comprises the following steps: freezing and grinding to prepare a test solution; preparing a control sample solution; and detecting illegal addition of chemical components in the sample by using a high performance liquid chromatography-tandem mass spectrometry method. The method is rapid, simple and convenient, has high sensitivity, and is suitable for qualitative screening and quantitative detection of illegally added 35 chemical components in the pediatric cough and asthma relieving patch of medical instruments.

Description

Method for determining 35 chemical components illegally added in patch for relieving cough and asthma of children

Technical Field

The invention relates to the technical field of drug detection, in particular to a method for determining 35 chemical components illegally added in a patch for relieving cough and asthma of children.

Background

At present, the children cough and asthma relieving patch products in the market of China mainly comprise three types of medicines, medical instruments and non-medicines with character strengthening numbers. The patch has the advantages of convenient use, small side effect, good compliance of children and the like, and becomes the first choice for relieving cough and asthma of many consumers.

At present, national standards and some documents stipulate or discuss illegal addition of chemical components in Chinese patent medicines for relieving cough and asthma and health-care food, but related compounds have single category or few categories detected at one time and cannot meet the requirement of rapid and comprehensive detection; and because the pretreatment of the viscous rubber matrix in the patch is complex, the medicine is not easy to extract, the detection of the added medicine has certain difficulty, and the detection method for illegally adding chemicals in the plaster medical apparatus is relatively rare in domestic and foreign documents.

Disclosure of Invention

In order to solve the problem in the prior art, the invention provides a method for determining 35 chemical components illegally added into a pediatric cough and asthma relieving patch, a sample is processed by a freeze grinding method, and a determination method of 35 cough and asthma relieving medicines is established by utilizing high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), so that reliable technical guarantee is provided for the illegal addition of the pediatric cough and asthma relieving patch in the medical instruments.

The invention is realized by the following technical scheme:

a method for determining 35 chemical components illegally added in a patch for relieving cough and asthma of children comprises the following steps:

(1) preparing a test solution: taking a cough and asthma relieving patch sample 5, sticking, removing an anti-adhesion layer, placing in a freezing grinding tube, adding a steel impactor into the grinding tube, introducing liquid nitrogen, driving the steel impactor to oscillate and impact a sample by a battery at-196 ℃, wherein the speed of oscillating and impacting the sample by the steel impactor is 10cps, the speed of oscillating and impacting the sample by the steel impactor is 2min each time, circulating for 2 times to obtain sample powder, quickly transferring the sample powder into 50mL of methanol, carrying out ultrasonic treatment for 20min, centrifuging for 10 min at 4000r/min, taking supernate, filtering by using a 0.22 mu m microporous membrane, and taking a subsequent filtrate to obtain the cough and asthma relieving patch;

(2) preparation of control sample solution: taking 10mg of 35 reference substances respectively, placing the reference substances into 10mL measuring bottles respectively, adding methanol for dissolving and diluting to the scale, shaking up, preparing into 1mg of sample mL respectively^-1The control stock solution of (a); precisely measuring 1mL of each reference substance stock solution, placing in a 100mL measuring flask, adding methanol to dissolve and dilute to scale, shaking to obtain 10 μ g of each reference substance^-1The mixed solution is gradually diluted by methanol until the concentration is respectively 20, 50, 100, 200, 500 and 1000ng mL^-1Mixed reference solution of (1); the control sample is codeine phosphate, dextromethorphan hydrobromide, dioxopromazine hydrochloride, benproperine phosphate, noscapine, ambroxol hydrochloride, bromhexine hydrochloride, salbutamol, ephedrine hydrochloride, terbutaline sulfate, procaterol hydrochloride, tulobuterol hydrochloride, clenbuterol hydrochloride, bamterol hydrochloride, chlorphenamine hydrochloride, dyphylline hydrochloride, doxofylline, dexamethasone, triamcinolone acetonide, cortisone acetate, clobetasol propionate, beclomethasone propionate, sulfamethoxazole, pipemidic acid, trimethoprim, sulfadiazine, ofloxacin, clarithromycin, roxithromycin, diazepam, nitrazepam, chlorpheniramine maleate, diphenhydramine hydrochloride, ketotifen fumarate, diclofenac sodium;

(3) and detecting illegal addition of chemical components in the sample by using a high performance liquid chromatography-tandem mass spectrometry method.

Wherein the chromatographic conditions of the high performance liquid chromatography are as follows: chromatographic column C18, column length 150mm, column internal diameter 3.0mm, filler particle size 2.7 μm; 0.01mol of mobile phase^-10.1% formic acid in aqueous ammonium acetate-acetonitrile; the gradient elution conditions were: 0-5 min, 10% acetonitrile; 5-12 min, 10% → 70% acetonitrile; 12-17 min, 70% → 100% acetonitrile; 17-20 min, 100% acetonitrile; 20-22 min, 100% → 10% acetonitrile; 22-27 min, 10% acetonitrile; the column temperature is 35 ℃, the flow rate is 0.5mL for min^-1The amount of sample was 1. mu.L. The high performance liquid chromatography conditions can ensure that chromatographic peaks of all components in the reference substance and the test solution achieve good separation effects.

Conditions for mass spectrometry were: the electrospray ion source is ESI; the detection mode is multi-reaction detection; the scanning mode is as follows: diclofenac sodium adopts a negative ion mode, other compounds adopt a positive ion mode, the atomization air pressure is 65psi, and the auxiliary air pressure is 65 psi; the positive ion mode adopts a spraying voltage of 5500V and a temperature of 550 ℃; the negative ion mode adopts the spraying voltage of-4500V and the temperature of 450 ℃.

Has the advantages that:

according to the method for determining the illegal addition of 35 chemical components in the patch for relieving cough and asthma of children, provided by the invention, the chemical components in the patch can be effectively extracted by a freeze grinding method in a pretreatment condition, the operation is simple, the extraction time is short, the extraction rate is high, and the use of chemical reagents is less; simultaneously adopting a high performance liquid chromatography-tandem mass spectrometry method, and taking C as the reference₁₈The chromatographic column is an analytical column, ammonium acetate aqueous solution containing 0.1% formic acid-acetonitrile is used as a mobile phase, gradient elution is carried out, and 35 illegal additive components in the patch can be separated and detected in an electrospray ion source (ESI) scanning mode and a multi-reaction monitoring (MRM) scanning mode. The linear relation of the 35 chemical components in the corresponding measuring range is good, and the correlation coefficients are all larger than 0.99; the recovery rate of the method is 80.0-120.0% (n = 9), the detection limit is 0.19-1.60 n g as mL^-1The limit of quantification is 0.62-5.35 ng as mL^-1. In 8 detected samples, diphenhydramine hydrochloride is detected in 1 sample, and the content of each patch is 0.8 mg.

In the development of the method, the selection of pretreatment, the selection of a mobile phase system and the setting of gradient elution play an important role in the accuracy and specificity of the method. The established method for measuring the 35 compounds can quickly and accurately measure chemical components illegally added in the pediatric cough and asthma relieving patch of the medical apparatus and instruments, and provides powerful support for fighting the fake behavior of the patch.

Drawings

FIG. 1 is a spectrum of codeine phosphate;

FIG. 2 is a spectrum of dextromethorphan hydrobromide;

FIG. 3 is a spectrum of dioxopromethazine hydrochloride;

FIG. 4 is a spectrum of benproperine phosphate;

FIG. 5 is a spectrum of narcotine;

FIG. 6 is a spectrum of ambroxol hydrochloride;

FIG. 7 is a spectrum of bromhexine hydrochloride;

FIG. 8 is a spectrum of salbutamol;

FIG. 9 is a spectrum of ephedrine;

FIG. 10 is a spectrum of terbutaline sulfate;

FIG. 11 is a spectrum of procaterol hydrochloride;

FIG. 12 is a spectrum of tulobuterol hydrochloride;

FIG. 13 is a spectrum of clenbuterol hydrochloride;

FIG. 14 is a spectrum of Banpedol hydrochloride;

FIG. 15 is a spectrum of chlorpropaline hydrochloride;

FIG. 16 is a spectrum of diprophylline;

FIG. 17 is a spectrum of doxofylline;

FIG. 18 is a spectrum of dexamethasone;

figure 19 is a spectrum of triamcinolone acetonide;

FIG. 20 is a spectrum of cortisone acetate;

FIG. 21 is a spectrum of clobetasol propionate;

FIG. 22 is a spectrum of beclomethasone dipropionate;

FIG. 23 is a spectrum of sulfamethoxazole;

FIG. 24 is a spectrum of pipemidic acid;

FIG. 25 is a spectrum of trimethoprim;

FIG. 26 is a spectrum of sulfadiazine;

FIG. 27 is a spectrum of ofloxacin;

FIG. 28 is a spectrum of clarithromycin;

FIG. 29 is a spectrum of roxithromycin;

figure 30 is a spectrum of diazepam;

figure 31 is a spectrum of nitrazepam;

FIG. 32 is a spectrum of chlorpheniramine maleate;

FIG. 33 is a spectrum of diphenhydramine hydrochloride;

FIG. 34 is a spectrum of ketotifen fumarate;

FIG. 35 is a spectrum of diclofenac sodium.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

1. Apparatus and materials

LC-MS: shimadzu LC-20AD liquid phase, American AB company API4000+ mass spectrometer; XSE-205DU model electronic balance: an allegory weight of 0.0001g, Mettler-Tollido, Switzerland; 6875 type A cryo-mill, SPEX Sample PREP, USA; ST16R model centrifuge, seimer feishel, usa. Codeine phosphate (batch No. 171203-, the content is as follows: 100.0%), clenbuterol hydrochloride (batch number: 100072-201503, content: 90.5%), bamotero hydrochloride (batch No.: 101009 and 200701, the content: 99.7%), chlorpropaline hydrochloride (batch No.: 100220 201603, content: 99.9%), diprophylline (batch number: 100417 201603, content: 99.9%), doxofylline (batch number: 100625-201202, content: 99.9%), dexamethasone (batch No.: 100129-: 99.8%), triamcinolone acetonide (batch No.: 100055-201804, content: 98.8%), cortisone acetate (batch No.: 100123-: 99.1%), clobetasol propionate (batch No.: 100302 and 201804, wherein the contents are as follows: 99.2%), beclomethasone dipropionate (batch number: 100119-201805, content: 98.8%), sulfamethoxazole (batch number: 100025-201505, content: 99.6%), pipemidic acid (batch No.: 100151 and 201904, wherein the contents are as follows: 84.3%), trimethoprim (batch No.: 100031-201606, content: 99.8%), sulfadiazine (batch number: 100026-: 99.7%), ofloxacin (batch No.: 130454-202007, content: 99.7%), clarithromycin (batch No.: 130558-: 98.6%), roxithromycin (batch No.: 130557-: 96.0%), diazepam (batch No.: 171225 and 201304, wherein the contents are as follows: 99.9%), nitrazepam (batch No.: 171217-: 99.9%), chlorpheniramine maleate (batch number: 100047-201507, content: 99.7%), diphenhydramine hydrochloride (batch No.: 100066-: 99.9%), ketotifen fumarate (batch No.: 100230 and 201904, the contents: 99.8%), diclofenac sodium (batch number: 100334-: 100.0 percent), were purchased from the institute for food and drug testing, 8 samples were all commercially available, methanol, acetonitrile, formic acid were all chromatographically pure, and ammonium acetate was analytically pure.

2. Method and results

2.1 liquid chromatography conditions

Column C18 (150 mm. times.3.0 mm, SB-Aq2.7 μm) (Agilent, USA); 0.01mol of mobile phase^-1Ammonium acetate (containing 0.1% formic acid)Carrying out gradient elution on the water solution-acetonitrile for 0-5 min, and carrying out 10% acetonitrile; 5-12 min, 10% → 70% acetonitrile; 12-17 min, 70% → 100% acetonitrile; 17-20 min, 100% acetonitrile; 20-22 min, 100% → 10% acetonitrile; 22-27 min, 10% acetonitrile; the column temperature is 35 ℃, the flow rate is 0.5mL for min^-1The amount of sample was 1. mu.L.

2.2 Mass Spectrometry conditions

Electrospray ion source (ESI); the detection mode is multi-reaction detection (MRM); the scanning mode is a positive ion mode and a negative ion mode (diclofenac sodium adopts the negative ion mode, other compounds adopt the positive ion mode), the atomization air pressure is 65psi, and the auxiliary air pressure is 65 psi. The positive ion mode adopts a spraying voltage of 5500V and a temperature of 550 ℃; the negative ion mode adopts the spraying voltage of-4500V and the temperature of 450 ℃.

2.3 preparation of control solutions

Taking about 10mg of each of 35 reference substances, precisely weighing, respectively placing in a 10mL measuring flask, adding methanol to dissolve and dilute to scale, shaking up, preparing into about 1mg of sample^-1Control stock solution of (4). Precisely measuring 1mL of each reference substance stock solution, placing in a 100mL measuring flask, adding methanol to dissolve and dilute to scale, shaking to obtain samples containing reference substances each containing 10 μ g of each sample^-1The mixed solution is gradually diluted by methanol until the concentration is respectively about 20, 50, 100, 200, 500 and 1000ng^-1Mixed control solution of (4).

2.4 preparation of test solutions

And (3) taking a sample 5, removing the non-stick layer, placing the sample in a freezing grinding tube, adding a steel impactor into the grinding tube, introducing liquid nitrogen, driving the steel impactor to vibrate and impact the sample (the speed is 10cps, each time is 2min, and the circulation is 2 times) by a battery at the temperature of-196 ℃ to obtain sample powder, quickly transferring the sample powder into 50mL of methanol, carrying out ultrasonic treatment for 20min, centrifuging for 10 min at the speed of 4000r/min, taking supernatant, filtering by using a 0.22 mu m microporous filter membrane, and taking a subsequent filtrate to obtain the product. When the concentration of the chemical component in the test solution is out of the linear range of the standard curve, the test solution is diluted to the linear range by methanol and then analyzed.

2.5 optimization of Mass Spectrometry conditions

Respectively collecting 35 reference substance stock solutions (about 1 mg) as well as mL^-1) Diluted by methanol to about 1 mu g and mL^-1Selecting an ESI ionization mode, carrying out single-needle sample injection, obtaining an excimer ion peak by adopting first-stage mass spectrum scanning, and optimizing the de-clustering voltage and the collision voltage of the ion generated by the molecular ion, wherein the ion with the highest abundance is used as a quantitative ion, and the ion with the second highest abundance is used as a qualitative ion. And simultaneously scanning the detected ions of each chemical component by adopting a multi-reaction monitoring (MRM) scanning mode under the condition of the optimal declustering voltage and collision voltage. The mass spectrum parameters of each chemical composition are shown in table 1.

TABLE 1 Mass Spectrometry parameters

Note: is a quantitative ion

2.6 methodological investigation

2.6.1 Linear relationship investigation

The concentration of the series under the item of 2.3 is 20, 50, 100, 200, 500 and 1000ng mL^-1Mixed control solution of (1), an "2.1 'and 2.2' in terms of chromatogram and mass spectrum, taking the peak area of the chromatographic peak of the quantitative ion pair of 35 chemical components as the ordinate (Y) and the concentration as the abscissa (X, ng) to be involved in mL^-1) And drawing a standard curve to obtain a linear regression equation, wherein the result is shown in a table 2. The results show that the linear relation between the concentration and the peak area is good in the specified range, and the correlation coefficients are all larger than 0.99.

2.6.2 specificity test

200ng mL of the concentration under the item of 2.3^-1The mixed reference substance solution is measured according to the conditions of '2.1' and '2.2', and the MRM chromatogram of the reference substance is obtained and is shown in figure 1; a blank sample is taken and processed and measured according to the method under the item 2.4, and interference peaks of all chemical components are not seen in a spectrogram of the blank sample, which indicates that the method has good specificity.

2.6.3 precision test

200ng mL of the concentration under the item of 2.3^-1The results of the 6-time parallel measurement of the mixed control solutions of (1) and (2.2) are shown in table 2, and RSD of the peak areas of the respective components is 0.42% to 5.45% (n = 6), indicating that the precision of the method is good.

2.6.4 repeatability test

Taking 6 parts of blank sample, respectively and precisely adding a proper amount of mixed reference solution to ensure that the concentration of each component is about 200ng as much as mL^-1The components are processed and measured according to the method under the item 2.4, the results are shown in the table 2, the peak area RSD of each component is 1.73-6.39% (n = 6), and the method is good in repeatability.

2.6.5 recovery test

Taking 9 parts of blank samples, wherein 3 parts are taken as a group, adding 100 ng/mL, 200 ng/500 ng/mL into the blank samples respectively^-1The mixed control solution of (4) was treated and measured according to the method under "2.4" to calculate the recovery rate. The results are shown in table 2, and the recovery rate of each component ranges from 80.0% to 120.0% (n = 9), indicating that the sample recovery rate is better.

2.6.6 stability test

The results of measurement of 1 part of the solution under the term of "2.6.4" after being placed at room temperature for 0, 2, 4, 8 and 12 hours are shown in table 2, and the RSD of the peak area of each component is 0.87% to 5.31% (n = 5), indicating good stability.

2.6.7 detection limit and quantification limit

An appropriate amount of the mixed control solution was diluted with methanol and measured, and the detection limit of each chemical component was calculated with S/N =3 and the quantification limit of each chemical component was calculated with S/N =10, and the results are shown in table 2.

2.7 sample determination

8 batches of samples circulating in the market are detected, diphenhydramine hydrochloride is detected from 1 batch of samples, and the content of each patch is calculated to be 0.8 mg.

TABLE 235 chemical composition methodology test results

Note: the numbers 1 to 35 are the same as those in Table 1.

3. Discussion of the related Art

At present, the treatment modes of medical appliance patches reported in the literature mainly comprise two modes, namely, the patches are cut and then directly extracted by methanol or ethanol and the like, and the extraction rate of the method is low; secondly, the extraction is carried out after the treatment of chemical reagents such as ether, normal hexane and the like, the method is complex to operate, and the using amount of the chemical reagents is large; the test adopts a freezing and grinding method, the sample is quickly frozen and then ground into powder, the contact area between the sample and a solvent can be increased, the release of chemical components in the sample is facilitated, the operation is simple, the extraction time is short, the extraction rate is high, and the use of chemical reagents is less.

The mother ions and the daughter ions of each component of the mixed reference substance have specificity and do not interfere with each other. The retention time of ion chromatographic peaks in the test solution and the reference solution is compared to preliminarily judge the chemical components possibly contained in the test solution, and then the contained chemical components are further confirmed by extracting parent ions and characteristic fragment ions.

The 35 compounds selected in this test cover antitussives (codeine phosphate, dextromethorphan hydrobromide, dioxopromazine hydrochloride, benproperine phosphate, noscapine), expectorants (ambroxol hydrochloride, bromhexine hydrochloride), epinephrine (salbutamol, ephedrine hydrochloride, terbutaline sulfate, procaterol hydrochloride, tulobuterol hydrochloride, clenbuterol hydrochloride, bambuterol hydrochloride, chlorphenaline hydrochloride), antiasthmatic (dyphylline, doxofylline), glucocorticoid (dexamethasone, triamcinolone acetonide, cortisone acetate, clobetasol propionate, beclomethasone propionate), antibiotics (sulfamethoxazole, pipemidic acid, trimethoprim, sulfadiazine, ofloxacin, clarithromycin, roxithromycin), sedative hypnotics (diazepam, nitrazepam), antiallergic (chlorphenamine maleate, diphenhydramine hydrochloride, doxylamine hydrochloride, sulfadiazine, doxiflavine hydrochloride, bromhexine hydrochloride), and the like, Ketotifen fumarate), non-steroidal anti-inflammatory drugs (diclofenac sodium) and other clinical common drugs have definite curative effects and low price, are easy to add in the cough and asthma relieving patch, and can have adverse effects on the health of children after long-term use. The method for measuring 35 compounds established in the test can quickly and accurately measure chemical components illegally added in the pediatric cough and asthma relieving patch of medical instruments, and provides powerful support for fighting the fake behavior of the patch.

Claims (2)

1. A method for determining 35 chemical components illegally added in a patch for relieving cough and asthma of children is characterized by comprising the following steps:

(1) preparing a test solution: taking a cough and asthma relieving patch sample 5, sticking, removing an anti-adhesion layer, placing in a freezing grinding tube, adding a steel impactor into the grinding tube, introducing liquid nitrogen, driving the steel impactor to vibrate and impact the sample by a battery at-196 ℃ to obtain sample powder, quickly transferring the sample powder into 50mL methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging at 4000r/min for 10 minutes, taking supernatant, filtering with a 0.22 mu m microporous membrane, and taking a subsequent filtrate to obtain the cough and asthma relieving patch;

the conditions of the steel striker oscillating to impact the sample are as follows: the speed is 10cps, each time is 2min, and the circulation is 2 times;

(2) preparation of control sample solution: taking 10mg of 35 reference substances respectively, placing the reference substances into 10mL measuring bottles respectively, adding methanol for dissolving and diluting to the scale, shaking up, preparing into 1mg of sample mL respectively^-1The control stock solution of (a); precisely measuring 1mL of each reference substance stock solution, placing in a 100mL measuring flask, adding methanol to dissolve and dilute to scale, shaking to obtain 10 μ g of each reference substance^-1The mixed solution is gradually diluted by methanol until the concentration is respectively 20, 50, 100, 200, 500 and 1000ng mL^-1Mixed reference solution of (1); the reference substance is codeine phosphate, dextromethorphan hydrobromide, dioxopromazine hydrochloride, benproperine phosphate, noscapine, ambroxol hydrochloride, bromhexine hydrochloride, salbutamol, ephedrine hydrochloride, terbutaline sulfate, procaterol hydrochloride, tulobuterol hydrochloride, clenbuterol hydrochloride, bamterol hydrochloride, chlorphenamine hydrochloride, dyphylline hydrochloride, doxofylline, dexamethasone, triamcinolone acetonide, cortisone acetate, clobetasol propionate, beclomethasone propionate, sulfamethoxazole, pipemidic acid, trimethoprim, sulfadiazine, ofloxacin, clarithromycin, roxithromycin, diazepam, nitrazepam, chlorpheniramine maleate, diphenhydramine hydrochloride, ketotifen fumarate, diclofenac sodium;

(3) detecting illegal addition of chemical components in the sample by using a high performance liquid chromatography-tandem mass spectrometry method;

the chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column SB-Aq C18, the column length is 150mm, the column inner diameter is 3.0mm, and the filler particle size is 2.7 μm; 0.01mol of mobile phase^-10.1% formic acid in aqueous ammonium acetate-acetonitrile;

the conditions of the mass spectrometry are as follows: the electrospray ion source is ESI; the detection mode is multi-reaction detection; the scanning mode is as follows: diclofenac sodium adopts a negative ion mode, other compounds adopt a positive ion mode, the atomization air pressure is 65psi, and the auxiliary air pressure is 65 psi; the positive ion mode adopts a spraying voltage of 5500V and a temperature of 550 ℃; the negative ion mode adopts the spraying voltage of-4500V and the temperature of 450 ℃;

the illegal addition is codeine phosphate, dextromethorphan hydrobromide, dioxopromazine hydrochloride, benproperine phosphate, noscapine, ambroxol hydrochloride, bromhexine hydrochloride, salbutamol, ephedrine hydrochloride, terbutaline sulfate, procaterol hydrochloride, tulobuterol hydrochloride, clenbuterol hydrochloride, bamterol hydrochloride, chlorphenamine hydrochloride, dyphylline, doxofylline, dexamethasone, triamcinolone acetonide, cortisone acetate, clobetasol propionate, beclomethasone propionate, sulfamethoxazole, pipemidic acid, trimethoprim, sulfadiazine, ofloxacin, clarithromycin, roxithromycin, diazepam, nitrazepam, chlorpheniramine maleate, diphenhydramine hydrochloride, ketotifen fumarate and diclofenac sodium.

2. The method for determining the 35 chemical components illegally added into the patch for relieving cough and asthma of children according to claim 1, wherein the gradient elution conditions of the high performance liquid chromatography are as follows: 0-5 min, 10% acetonitrile; 5-12 min, 10% → 70% acetonitrile; 12-17 min, 70% → 100% acetonitrile; 17-20 min, 100% acetonitrile; 20-22 min, 100% → 10% acetonitrile; 22-27 min, 10% acetonitrile; the column temperature is 35 ℃, the flow rate is 0.5mL for min^-1The amount of sample was 1. mu.L.

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