I believe that antibiotics are familiar to everyone. As an anti-inflammatory drug, it is widely used in the treatment of various diseases, but the harm of abuse is also obvious; and in recent years, in addition to European and American countries, antibiotics have been found in surface water. In addition to exceeding the standard, some scientific research and testing personnel in my country also found high levels of antibiotics when sampling and testing the Yangtze River and the Yellow River. Most of these antibiotics are discharged into surface water from medical wastewater, domestic sewage, aquaculture wastewater, animal drugs, feed, etc., and then some of them enter the human body through drinking water. This is also the reason why a number of testing standards for antibiotics in water have been introduced in recent years. Today we will talk about the method of liquid chromatography-triple quadrupole mass spectrometry for the detection of antibiotics in water.
The detection principle of this method is to filter the collected water samples through a 0.22 pm filter, mix them with acetonitrile 1:1, and then inject the samples, and then use a liquid chromatography-triple quadrupole mass spectrometer for determination. According to retention time and characteristic ion qualitative, external standard method for quantification.
Reagents used for testing
1. Acetonitrile: chromatographically pure.2. Formic acid: chromatographically pure.
4. 50% acetonitrile solution: take 50 mL of acetonitrile and dilute to 100 mL with water.
5. 0.1% formic acid aqueous solution: take 1 mL of formic acid and dilute to 1000 mL with water.
6. Standard product: erythromycin, content ≥ 97.0%; penicillin, content ≥ 98.0%.
7. Erythromycin standard stock solution, the concentration is 100ug/mL. Accurately weigh 10.0 mg of erythromycin standard, dissolve and dilute it with acetonitrile into a standard stock solution with a concentration of 100ug/mL; you can also directly purchase certified standard solutions, freeze at -10°C, and store in the dark; or refer to the manufacturer Store according to the manufacturer's product instructions, and should be returned to room temperature and shaken well before use.
8. Penicillin standard stock solution, the concentration is 100ug/mL. Accurately weigh 10.0 mg of penicillin standard, dissolve and dilute with 50% acetonitrile solution into a standard stock solution with a concentration of 100ug/mL; you can also directly purchase certified standard solutions, and store them in a freezer below -10°C and in the dark; or refer to Store according to the manufacturer's product instructions, bring to room temperature and shake well when using.
9. Mixed standard solution of erythromycin and penicillin: the concentration is 10.0ug/mL. Take 1.00 mL of the standard stock solutions of erythromycin and penicillin respectively in a 10.0 mL volumetric flask, and dilute with 50% acetonitrile solution to obtain the mixed standard solution of erythromycin and penicillin.
10. Filter: Disposable syringe filter, 0.22um, made of mixed cellulose ester (MCE).
11. Disposable polypropylene syringe.
Instruments used for testing
1. Liquid chromatography-triple quadrupole mass spectrometer: equipped with electrospray ionization (ESI), with gradient elution and multiple reaction monitoring functions.2. Analytical balance: the inductive amount is 0.0001g.
3. Sample bottle: 100mL ground mouth or amber glass bottle with teflon inner liner cap.
4. Micro syringe or pipette: 10ul, 50ul, 100ul, 500ul, 1.0mL.
Collection and preparation of water samples
We can collect water samples according to relevant regulations. The sample bottle should be filled with no gaps when collecting the water sample. Refrigerate below 4°C and store in the dark. The analysis was completed within 2 days.Preparation of water samples
The water sample needs to be thoroughly mixed, take about 10mL of the sample and filter it through a 0.22um filter membrane, discard at least 1mL of the primary filtrate, accurately pipette 500uL of the filtered sample into a brown injection bottle, add 500uL of acetonitrile, and wait for a while after mixing. Measurement.
Preparation of blank water samples
Replace the water sample with laboratory grade pure water, and then follow the water sample preparation steps for the blank water sample.
Detection steps
Liquid chromatography conditionsThe chromatographic column is C18 (100mmX2.1mm, particle size is 1.7um), or equivalent; the mobile phase is A: acetonitrile, B: 0.1% formic acid aqueous solution; the flow rate is 0.25mL/min; the column temperature is 30°C; the injection volume It is 10uL; the mobile phase gradient elution procedure can refer to the corresponding chart.
MS conditions
The ion source is electrospray ion source (ESI); the scanning mode is positive ion mode; the monitoring mode is multiple reaction monitoring (MRM); the desolvation gas, cone gas, and collision gas are all high-purity nitrogen or other suitable gases;
Establishment of standard curve
Pipette an appropriate amount of mixed standard solution of erythromycin and penicillin, and dilute it with 50% acetonitrile solution to prepare standard series solutions with at least 5 concentration points. The reference concentration of erythromycin and penicillin in the standard solution is 0.100ug/ L, 0.200ug/L, 0.500ug/L, 1.00ug/L, 2.00ug/L, 5.00ug/L, 10.0ug/L and 20.0ug/L, pipette 1.0mL of standard series solution into a brown injection bottle , to be tested. According to the specified instrument conditions, the standard series of solutions were measured in turn from low concentration to high concentration. Take the mass concentration (ug/L) of the target compound as the abscissa and its corresponding response value as the ordinate to establish a standard curve.
Determination of water samples
The prepared water samples were measured according to the specified instrument conditions.Qualitative analysis can measure the standard solution and sample under the same chromatographic/mass spectrometry conditions, according to the retention time of the standard solution chromatogram and the retention time of the sample, the characteristic ions of each chromatographic peak of the standard solution and the characteristics of each chromatographic peak of the sample Ion contrast qualitative. The relative deviation of the retention time between the sample and the reference material is not more than 5%. The relative abundance (Ksam) of the qualifier product ion of the target compound in the sample. Compared with the relative abundance (Kskd) of the corresponding qualifier ion in the standard solution with similar concentration, if the relative abundance deviation does not exceed the range specified in the relevant chart, it can be determined that the corresponding target compound exists in the sample.
The mass concentration of erythromycin and penicillin in the water sample can be calculated directly according to the formula.
