CN113237983B - Solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method for oxypurinol in water - Google Patents
Solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method for oxypurinol in water Download PDFInfo
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Abstract
A solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method for water quality oxypurinol belongs to the technical field of analysis of water quality oxypurinol. The method has the advantages of high analysis speed, accurate qualitative determination, high sensitivity, good stability, strong operability and easy popularization and use.
Description
Technical Field
The invention belongs to the technical field of analysis of water quality oxypurinol, and particularly relates to a solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method of water quality oxypurinol.
Background
Oxypurinol (OXY) is the main active substance of allopurinol, which is listed in the basic drug list of the World Health Organization (WHO) and is widely used in the treatment of gout and hyperuricemia in many countries. Allopurinol is metabolized to OXY 80% in the human body. OXY, one of the Active Pharmaceutical Ingredients (APIs), can enter the environment by the following routes: excretion by drug users, improper disposal of unused drugs, discharge from manufacturing processes, and municipal sewage treatment plants. OXY can be present in rivers, precipitation and groundwater because of its apparently high biopersistence and polarity, whereas only very low concentrations of allopurinol are detected in the raw wastewater of sewage treatment plants despite the high prescribed amount of allopurinol. There is also evidence that OXY is stable in wastewater treatment processes, with wastewater concentrations at the wastewater treatment plant inlet and outlet of 26.6. Mu.g/L and 21.7. Mu.g/L, respectively. Stable and refractory OXY poses, like other active pharmaceutical ingredients, a number of threats to the aquatic ecosystem, wildlife and plant and human body, such as common human toxicity, ecotoxicity and drug resistance.
At present, a solid phase extraction/ultra-high performance liquid chromatography-fluorescence detection method (SPE/UHPLC-FLD) for water quality OXY is not established in China, so that the development of a sensitive and reliable method suitable for trace OXY detection is urgent, so as to meet the urgent needs of environment monitoring work of newly-developed organic pollutants OXY in water quality in China, such as the development of source and risk assessment of the OXY pollutants in an environmental water body, the monitoring and assessment of the OXY risk distribution, the development of OXY ecological and environmental impact assessment, the implementation of OXY pollution prevention and control, limitation measures and the like.
Disclosure of Invention
The invention aims to provide a solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method of water quality oxypurinol aiming at the defects of the prior art.
And enriching the oxypurinol in the water by adopting a solid phase extraction technology, eluting by using methanol, concentrating eluent, and separating and detecting by using an ultra-high performance liquid chromatograph with a fluorescence detector. Qualitative according to retention time, quantitative by external standard method.
When the volume of the enriched sample is 200ml, the detection limit of the method is 1.30. Mu.g/L, and the lower limit of the determination is 5.20. Mu.g/L.
The advantages are that:
the principle of the solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method of the water quality oxypurinol is that after a water sample is subjected to solid-phase extraction, the oxypurinol in the water is separated by a liquid chromatograph, detected by a fluorescence detector, qualitative according to the retention time, and quantitative by an external standard method. The method obtains the ultrasensitive fluorescent signal response of the oxypurinol on the ultra-high performance liquid chromatography, and realizes the rapid, accurate and trace analysis of the oxypurinol within 1.5 minutes.
The method has the advantages of high analysis speed, accurate qualitative determination, high sensitivity, good stability, strong operability and easy popularization and use. The development and establishment of the detection method have very important significance for promoting the development of the environmental monitoring method in China, and are more beneficial to the full-force complementary environmental-friendly short-plate attack and solidness battle with the improvement of the environmental quality as the core.
The method obtains the ultrasensitive fluorescent signal response of the oxypurinol on the ultra-high performance liquid chromatography, and realizes the rapid, accurate and trace analysis of the oxypurinol.
The indexes of the method, such as detection limit, precision, accuracy and the like, all reach the advanced level at home and abroad at present, and the method and the system have very important significance for establishing a monitoring technology method and a system of emerging pollutants for the technology.
The establishment of the method has important significance for developing the source and risk assessment of new pollutants in environmental water, monitoring and assessing the risk distribution of the oxypurinol, developing the ecological and environmental impact assessment of the oxypurinol, implementing the oxypurinol pollution prevention and control and limitation measures and the like.
Drawings
FIG. 1 is a standard sample chromatogram of oxypurinol.
Detailed Description
2.1 principle of method:
the solid phase extraction technology is adopted to enrich the oxypurinol in the water, the oxypurinol is eluted by methanol, and after the eluent is concentrated, the ultrahigh performance liquid chromatograph with a fluorescence detector is used for separation and detection. Qualitative according to retention time, quantitative by external standard method.
2.2 reagents and materials:
the reagents used in the method are all analytically pure chemical reagents meeting the national standard except for other notes. Unless otherwise indicated, all water referred to in the present process is distilled water free of organic matter.
2.2.1 methanol (CH) 3 OH): and (4) liquid chromatography purification.
2.2.2 Oxypurinol Standard solution: ρ =1000 μ g/ml, a commercially available certified standard solution can be purchased. The standard solution was refrigerated below 4 ℃.
2.2.3 Oxyiurinol Standard stock solutions: ρ =100 μ g/ml, 1.00ml of the oxypurinol standard solution (2.2.2) was transferred to a 10ml volumetric flask and diluted to the mark with methanol (2.2.1). The standard stock solution was refrigerated below 4 ℃.
2.2.4 Oxypurinol Standard solutions: ρ =1.00 μ g/ml, 1.00ml of an oxypurinol standard stock solution (2.2.2) was transferred to a 100ml volumetric flask and diluted to the mark with methanol (2.2.1). The standard use solution is refrigerated below 4 ℃.
2.2.5hlb solid phase extraction column: 200mg/6mL. (HLB hydrophilic lipophilic balance).
2.2.6 of nitrogen with the purity more than or equal to 99.999 percent and is used for drying and concentrating samples.
2.3 instruments and devices:
2.3.1 ultra high performance liquid chromatograph: has fluorescence detector and gradient elution function.
2.3.2 chromatography column: the filler is 1.8 μm HSS T3 (octadecylsilane chemically bonded silica), the column length is 25cm, and the inner diameter is 50 mm.
2.3.3 sampling bottle: 500ml brown glass vial with ground stopper.
2.3.4 concentration plant: rotary evaporation device or concentrator.
2.3.5 solid phase extraction device.
2.3.6 needle Filter (Millipore): 0.22 μm, organic phase resistant.
2.3.7 general laboratory instruments.
2.4 sample:
2.4.1 sample Collection:
and collecting and storing samples according to related regulations of HJ/T164 technical Specifications for monitoring underground water environment and HJ/T91 technical Specifications for monitoring surface water and sewage. The sample must be collected in a pre-washed and dried sampling bottle (2.3.3), which cannot be pre-washed with a water sample prior to sampling to prevent contamination or adsorption of the sample. The sampling bottle is completely filled without air bubbles.
2.4.2 preservation of samples:
after the sample is collected, the sample should be refrigerated under the condition of keeping away from light and at the temperature of below 4 ℃, and the analysis is finished within 7 days.
2.5 analysis step:
2.5.1 solid phase extraction:
2.5.1.1 the HLB solid phase extraction column (2.2.5) was mounted on the solid phase extraction device (2.3.5).
2.5.1.2 activated solid phase extraction column: the HLB solid phase extraction column (2.2.5) was activated with 5ml of methanol (2.2.1) to make the solvent flow clean. The HLB solid phase extraction column (2.2.5) was then activated with 5ml of experimental water. During the activation process, the column head of the solid phase extraction column is ensured to be soaked, and the column is not allowed to run dry.
2.5.1.3 enrichment of samples: the sample is placed to the room temperature, 200ml is accurately measured (the volume of the water sample used for enrichment can be properly increased and decreased according to the water quality condition), and the water sample flows through the activated HLB solid-phase extraction column at the flow rate of 3-5 ml/min.
2.5.1.4 rinsing: eluting the enriched HLB solid-phase extraction column with 5ml of experimental water, eluting the impurities which are remained on the solid-phase extraction column, and discarding the eluent.
2.5.1.5 elution: the washed HLB solid phase extraction column was eluted with 5ml of methanol (2.2.1), and the methanol eluate was received by a receiving tube.
2.5.1.6 concentration: concentrating the methanol eluate to less than 1.0ml with a concentrating device (2.3.4), adding methanol (2.2.1) to accurately volume to 1.0ml, filtering with 0.22 μm needle filter (2.3.6), placing in brown sample bottle, and determining. If the concentration of the sample is too high, the sample can be diluted properly (the dilution factor can be obtained by a technician according to the requirements of industry standards).
2.5.2 chromatographic conditions:
mobile phase: 5% methanol +95% water (V: V).
Flow phase flow rate: 0.4ml/min.
Wavelength of fluorescence detector: the excitation wavelength λ ex was 254nm and the emission wavelength λ em was 359nm.
Sample introduction amount: 5 μ l.
2.5.3 plotting of Standard Curve:
a standard series of at least 5 concentration points was prepared in methanol using an amount (sufficient to use) of an oxypurinol standard use solution (2.2.4) at 5.00. Mu.g/L, 10.0. Mu.g/L, 20.0. Mu.g/L, 50.0. Mu.g/L and 100. Mu.g/L by mass, respectively, and stored in brown vials. Taking 5 mul of each concentration, performing ultra performance liquid chromatography analysis, and drawing a standard curve by taking the peak height or peak area as a vertical coordinate and the concentration as a horizontal coordinate. The correlation coefficient of the standard curve should be > 0.999, otherwise the standard curve is redrawn.
2.5.4 chromatogram of standard sample see FIG. 1:
2.5.5 continuous calibration:
the standard curve should be checked by measuring the solution at the middle point of the curve on each working day. If the relative deviation is less than or equal to 10%, the standard curve can still be used, otherwise, a new standard curve is drawn.
2.5.6 determination of samples
5 mul of sample to be measured is injected into an ultra high performance liquid chromatograph. The retention time and peak height (or peak area) of the chromatographic peak are recorded.
2.5.7 blank test:
when the sample is analyzed, a blank test is carried out, namely distilled water is used for replacing a water sample, the analysis is carried out according to the same determination steps as the sample, and the blank test result is lower than the detection limit of the method.
2.6 results calculation:
2.6.1 qualitative analysis:
characterized by the retention time of the target compound.
2.6.2 results calculation:
the concentration of oxypurinol in the sample (μ g/L) was calculated according to the formula (1):
in the formula:
ρ x -the mass concentration of oxypurinol in the sample, μ g/L.
ρ 1 The mass concentration of the olanexidine, μ g/L, was found from the standard curve (2.5.3).
V i The volume of the solid phase extract after concentration, ml.
D is dilution multiple.
V-water sample volume, ml.
2.6.3 results show:
the measurement results retain three significant digits.
2.7 precision and accuracy:
respectively selecting a blank sample, surface water and an actual wastewater sample, performing a matrix labeling experiment according to all steps of sample analysis, determining the content of the oxypurinol, performing parallel determination on each sample for 6 times, and calculating the relative standard deviation and the recovery rate, wherein the specific results are shown in tables 1 and 2. The relative standard deviation of the experimental analysis and determination result is 3.1-6.0%, and the recovery rate of the added standard is 90.1-99.5%.
TABLE 1 determination of surface Water precision and recovery by adding standard
TABLE 2 wastewater precision and recovery with standard
Note: no target was detected in any of the actual samples.
Claims (3)
1. A solid phase extraction/ultra high performance liquid chromatography-fluorescence detection method of water quality oxypurinol is characterized by comprising the following steps:
enriching the oxypurinol in water by adopting a solid phase extraction technology, eluting by using methanol, concentrating eluent, separating and detecting by using an ultra-high performance liquid chromatograph with a fluorescent detector, and performing qualitative detection according to retention time and quantitative detection by using an external standard method;
1) Solid phase extraction:
1.1 Mounting the HLB solid-phase extraction column on a solid-phase extraction device;
1.2 ) activating a solid phase extraction column: activating the HLB solid phase extraction column with methanol to make the solvent flow clean; activating the HLB solid-phase extraction column by using experimental water;
1.3 Enrichment of the sample: the water sample is placed to the room temperature and flows through the activated HLB solid-phase extraction column at the flow speed of 3-5 ml/min;
1.4 ) leaching: eluting the enriched HLB solid-phase extraction column with experimental water, eluting the impurities remained on the solid-phase extraction column, and discarding the eluate;
1.5 Elution): eluting the washed HLB solid phase extraction column by using methanol, and receiving the methanol eluent by using a receiving tube;
1.6 ) concentrating: concentrating the methanol eluent by a concentration device to be lower than 1.0ml, adding methanol to accurately fix the volume to 1.0ml, filtering, placing in a brown sample bottle, and determining;
2) Chromatographic conditions are as follows:
mobile phase: 5% methanol +95% water, volume ratio V: v;
flow rate of mobile phase: 0.4ml/min;
wavelength of fluorescence detector: the excitation wavelength λ ex is 254nm, and the emission wavelength λ em is 359nm;
sample introduction amount: 5 mu l of the solution;
3) Drawing a standard curve:
preparing a standard series of at least 5 concentration points by using an oxypurinol standard use solution in methanol, performing ultra high performance liquid chromatography analysis on each concentration, and drawing a standard curve by using peak height or peak area as a vertical coordinate and concentration as a horizontal coordinate;
4) Drawing a chromatogram of the standard sample;
5) And (3) determination of a sample:
injecting a sample to be detected into an ultra-high performance liquid chromatograph; recording the retention time and peak height, retention time and peak area of chromatographic peaks;
6) The concentration of oxypurinol in the sample (μ g/L) was calculated according to the formula (1):
in the formula:
ρ x -the mass concentration of oxypurinol in the sample, μ g/L;
ρ 1 -finding the mass concentration of the olceurinol, μ g/L, from the standard curve;
V i the volume of the concentrated solid phase extract, ml;
d is dilution multiple;
v-water sample volume, ml.
2. The solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection method of aqueous oxypurinol according to claim 1, characterized by comprising the following steps:
in the solid phase extraction step, if the concentration of the water sample is too high, dilution is performed.
3. The method for solid-phase extraction/ultra-high performance liquid chromatography-fluorescence detection of aqueous oxypurinol according to claim 1, which is characterized by comprising the following steps:
the filtration was performed through a 0.22 μm needle filter.
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