CN107478477B - Serum treatment method for jaundice, hemolysis and lipemia - Google Patents
Serum treatment method for jaundice, hemolysis and lipemia Download PDFInfo
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Abstract
The invention provides a method for treating serum of jaundice, hemolysis and lipemia, which comprises the steps of taking 100 mu L of the serum, adding 120 mu L0.2.2 mo L0/L1 perchloric acid and 80 mu L20.1.1 mo L/L glacial acetic acid, uniformly mixing, carrying out water bath at 37 ℃ for 10min, then adding 200 mu L0.25 mo L/L NaOH and 500 mu L0.2 mo L/L phosphate buffer solution, and then determining the endotoxin content through a bacterial endotoxin detection kit.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a serum treatment method for jaundice, hemolysis and lipemia.
Background
The variety of pyrogens for detecting medicines by a bacterial endotoxin detection method specified in Chinese pharmacopoeia of 2015 edition has more than 600. Meanwhile, the clinical application of the limulus reagent is gradually paid attention, the number of patients infected with gram-negative bacteria is large every year in the country, and most patients have the characteristics of high morbidity, quick disease progression, high mortality and the like after being infected with the gram-negative bacteria, so that the health of human beings is seriously threatened. The main component of the cell wall of gram-negative bacteria is lipopolysaccharide (bacterial endotoxin), and researchers at home and abroad find that the endotoxin level can be detected from body fluid or blood to provide an effective basis for diagnosing the gram-negative bacteria.
The bacterial endotoxin detection kit has been applied to most clinical cases for many years, and jaundice, hemolysis and lipemia are caused by a plurality of diseases. Jaundice causes an increase in serum bilirubin; hemolysis causes an increase in serum hemoglobin; lipemia causes elevation of triglycerides in serum. Bilirubin, hemoglobin, and triglycerides cause the serum to appear yellow, red, and turbid, respectively. The serum with the property affects result observation, and when the serum is detected by an instrument, the detected OD value is higher, and false positive may be generated. The three substances of bilirubin, triglyceride and heme also interfere with the normal reaction of limulus test. At present, a kit used by hospitals mostly adopts a heating dilution method to treat samples, but the method can not effectively treat jaundice samples, lipemia samples and hemolysis samples and is not suitable for treating the three special serum samples; in addition, it is difficult to completely remove the interfering substances in the serum (many proteases, blood coagulation factors, globulin, etc. exist in human serum) by the thermodilution method, and the accuracy of the detection is also affected, so that the detection result is false negative or false positive; secondly, since the chemical structure of endotoxin is composed of 3 distinct regions: o specific side chain, core polysaccharide and lipoid A, KDO bond is connected between the core polysaccharide and the lipoid, the lipoid A is glycolipid formed by lipidated glucosamine disaccharide through pyrophosphate, a serum sample is treated by a heating dilution method, and the heating can destroy the chemical structure of endotoxin, thereby influencing the detection result and possibly generating false negative phenomenon. In view of this, we repeatedly studied several serum treatment methods in order to eliminate the influence of these three types of bilirubin, hemoglobin, and triglyceride. Finally, the inventor finds that the method for treating the three jaundice, hemolysis and lipemia serum, namely the perchloric acid-glacial acetic acid method for treating the serum can eliminate the interference of the three substances (bilirubin, triolein and heme). The perchloric acid-glacial acetic acid serum treatment solution is repeatedly tested to develop a unique formula, and the components contain perchloric acid, glacial acetic acid, NaOH and KCl, and are used for the pretreatment of jaundice, hemolysis and lipemia samples, so that the interference of yellow, red and turbidity serum characters containing bilirubin, hemoglobin or triglyceride is effectively overcome, and the ideal recovery rate is obtained; meanwhile, a plurality of proteases, blood coagulation factors and globulins in serum can be removed, so that the protease interference in a limulus reagent test is reduced to the maximum extent, and the anti-interference capability is greatly improved; in addition, phosphate buffer solution is added in the preparation process of the perchloric acid-glacial acetic acid serum treatment solution, so that the product is uniform and stable, the endotoxin structure is protected to the maximum extent, and the endotoxin concentration is detected more accurately.
The principle of detecting bacterial endotoxin (L PS) by a bacterial endotoxin detection kit (end-point chromogenic substrate method) is that the bacterial endotoxin activates factor C in a main reagent, the activated factor C activates factor B, the activated factor B activates proclotting enzyme to form coagulase, the coagulase hydrolyzes a chromogenic substrate (Boc-L eu-Gly-Arg-pNA), so that pNA (yellow) is liberated, red azo pigment is formed after diazo coupling, and the bacterial endotoxin content can be determined by determining the absorbance at the wavelength of 545 nm.
Disclosure of Invention
The purpose of the present invention is to provide a serum treatment agent for jaundice, hemolysis, and lipemia.
In order to achieve the purpose, the invention adopts the following technical scheme:
a serum treatment liquid for treating jaundice, hemolysis and lipemia is characterized in that the treatment liquid comprises the following components of (1) 0.2mo L/L perchloric acid, 0.1mo L/L glacial acetic acid, (2) 0.25mo L/L NaOH, and (3) 0.2mo L/L phosphate buffer.
The 0.2mo L/L phosphate buffer solution is 0.2 mol/L potassium dihydrogen phosphate solution and 0.2 mol/L sodium hydroxide solution.
A method for treating icterus, hemolysis and lipemia with serum includes such steps as adding 120 mu L0.2 mo L0/L1 perchloric acid +80 mu L20.1.1 mo L/L glacial acetic acid to 100 mu L of serum, mixing, water bathing at 37 deg.C for 10min, adding 200 mu L0.25.25 mo L/L NaOH and 500 mu L0.2 mo L/L phosphate buffer, and measuring endotoxin content by bacterial endotoxin detecting kit.
The invention has the advantages that:
(1) the components of the treatment solution method of the invention contain perchloric acid, glacial acetic acid and sodium hydroxide, and phosphate buffer solution is added in the detection procedure, so that the stability is enhanced, the treatment solution is used for jaundice, hemolysis and lipemia samples, the interference of bilirubin, hemoglobin or triglyceride is effectively overcome, and the recovery rate is improved. Meanwhile, a plurality of proteases, blood coagulation factors and globulins in serum can be removed, so that the protease interference in a limulus reagent test is reduced to the maximum extent, and the anti-interference capability is greatly improved. In addition, phosphate buffer solution is added in the preparation process of the perchloric acid-glacial acetic acid serum treatment solution, so that the product is uniform and stable, the endotoxin structure is protected to the maximum extent, and the endotoxin concentration is detected more accurately.
(2) Compared with a perchloric acid-glacial acetic acid operation flow, the perchloric acid method mainly has the difference that the perchloric acid method needs water bath at 37 ℃ for two times, 15min and 3000rpm centrifugation for 10min, and the perchloric acid-glacial acetic acid method only needs water bath for one time, 10min and no centrifugation, so that the reaction time is shortened, the operation steps are simplified, and the perchloric acid-glacial acetic acid method is not easy to pollute.
Detailed Description
1. The formula comprises the steps of repeatedly testing and groping, pretreating jaundice, lipemia and hemolytic serum by using sulfuric acid, hydrochloric acid, nitric acid, perchloric acid and glacial acetic acid, finally selecting perchloric acid and glacial acetic acid as serum treatment liquid with the best effect according to repeated comparison anti-interference capability and recovery rate test results, and establishing the perchloric acid-glacial acetic acid serum treatment liquid as the following components of a formula (1) 0.2mo L/L perchloric acid, 0.1mo L/L glacial acetic acid, (2) 0.25mo L/L NaOH, and (3) 0.2mo L/L phosphate buffer solution (namely 0.2 mol/L dihydrogen phosphate solution and 0.2 mol/L sodium hydroxide solution).
2. The detection method comprises the following steps:
at present, the kit used in hospitals mostly adopts a heating dilution method to treat samples, and the main treatment process comprises the steps of taking serum of 100 mu L +900 mu L for examination, using water → 75 ℃ water bath for 10min → using a bacterial endotoxin detection kit to determine the content of endotoxin;
according to the existing method for pre-treating serum in the existing foreign documents, the main flow of the perchloric acid method is that 100 mu L +100 mu L0.18.18N NaOH is taken as serum, 5min at 37 ℃ of water bath → 100 mu L0.32N HClO4 → 10min at 37 ℃ of water bath → 200 mu L0.18.18 NNaOH → 500 mu L0.2N Tris-HCl (pH 8.0) → 3000rpm is centrifuged for 10min → a bacterial endotoxin detection kit is used for determining the endotoxin content;
the perchloric acid-glacial acetic acid method mainly comprises the following steps of taking serum (jaundice, lipemia and hemolysis) of 100 mu L +120 mu L0.2.2 mo L0/L1 HClO4+ 80 mu L20.1 mo L/L glacial acetic acid → 37 ℃ water bath for 10min → adding 200 mu L0.25 mo L/L NaOH +500 mu L0.2.2 mo L/L phosphate buffer → a bacterial endotoxin detection kit to determine the content of endotoxin.
Example 1
1 jaundice sample:
the clinical bilirubin concentration value in the serum of healthy people is 1mg/d L, the bilirubin concentration in the serum of general jaundice patients is about 20 times of the bilirubin concentration in the serum of healthy people, namely about 20mg/d L, a mathematical grade 10-time addition method is used in the test, and the test serum samples are 10 times, 20 times and 30 times of the bilirubin concentration value in the serum of normal people, namely the bilirubin concentration values are 10mg/d L, 20mg/d L and 30mg/d L.
1.1 interference bias test requirements
All instruments in direct contact with the reagents should be sterile and free of pyrogen contamination.
1.2 Experimental methods
Three test samples (control, bilirubin addition, bilirubin and endotoxin addition, four times per test sample) were prepared for each healthy population serum sample.
1.3 samples
The control sample is prepared by mixing 0.9m L serum sample (No. 1-6) +0.1m L normal saline.
Interference sample 1, 0.9m L serum sample of healthy population (No. 1-6) +0.1m L100 mg/d L bilirubin standard is mixed, and the addition concentration is 10mg/d L.
Sample 1 was recovered by taking 90 μ L interference sample (No. 1-6) +10 μ L0.500 EU/m L endotoxin solution → 100 μ L serum containing 0.050EU/m L endotoxin and 10mg/d L bilirubin.
Interference sample 2, 0.9m L serum sample of healthy population (No. 1-6) +0.1m L200 mg/d L bilirubin standard is mixed, and the addition concentration is 20mg/d L.
Sample 2 was recovered by taking 90 μ L interference sample (No. 1-6) +10 μ L0.500 EU/m L endotoxin solution → 100 μ L serum containing 0.050EU/m L endotoxin and 20mg/d L bilirubin.
Interference sample 3, the serum sample of 0.9m L healthy population (No. 1-6) +0.1m L300 mg/d L bilirubin standard is mixed, and the addition concentration is 30mg/d L.
Sample 3 was recovered by taking 90 μ L interference sample (No. 1-6) +10 μ L0.500 EU/m L endotoxin solution → 100 μ L serum containing 0.050EU/m L endotoxin and 30mg/d L bilirubin.
1.4 serum treatment method
The method comprises the following steps: heating dilution method
Control samples (No. 1-6), interference samples 1 (No. 1-6), recovery samples 1 (No. 1-6), interference samples 2 (No. 1-6), recovery samples 2 (No. 1-6), interference samples 3 (No. 1-6), and recovery samples 3 (No. 1-6) were examined by water → 75 ℃ water bath for 10min → determination of endotoxin content by bacterial endotoxin test kit, 100. mu. L + 900. mu. L, respectively.
The method 2 comprises the following steps: perchloric acid process
Control sample (Nos. 1 to 6), interference sample 1 (Nos. 1 to 6), recovery sample 1 (Nos. 1 to 6), interference sample 2 (Nos. 1 to 6), recovery sample 2 (Nos. 1 to 6), interference sample 3 (Nos. 1 to 6), and recovery sample 3 (No. 1 to 6) each 100. mu. L + 100. mu. L0.18.18N NaOH → 37 ℃ water bath 5min → 100. mu. L0.32N HClO4Water bath at 37 ℃ for 10min → 200 μ L0.18N NaOH → 500 μ L0.2N Tris-HCl (pH 8.0) → 3000rpm centrifugation for 10min → determination of endotoxin content by bacterial endotoxin detection kit.
The method 3 comprises the following steps: perchloric acid-glacial acetic acid process
Control sample (Nos. 1 to 6), interference sample 1 (Nos. 1 to 6), recovery sample 1 (Nos. 1 to 6), interference sample 2 (Nos. 1 to 6), recovery sample 2 (Nos. 1 to 6), interference sample 3 (Nos. 1 to 6), and recovery sample 3 (Nos. 1 to 6) each 100. mu. L + 120. mu. L0.2.2 mo L/L HClO4+80 μ L0.1 mo L/L glacial acetic acid → 37 ℃ water bath for 10min → 200 μ L0.25.25 mo L/L NaOH +500 μ L0.2 mo L/L phosphate buffer → bacterial endotoxin test kit to determine endotoxin content.
1.5 results (unit: EU/m L)
We summarize the following table based on the results of the interference and recovery tests:
(Note: general clinical experience data, serum bilirubin in jaundice is about 20 times higher than that in normal people, namely about 20mg/d L).
The conclusion is that when the bilirubin concentration of serum is within 10mg/d L, the serum is light yellow, at this time, when the bilirubin concentration is measured by a thermodilution method and a perchloric acid method, the interference deviation is up to 317%, the recovery rate is outside the interval of 50-200%, both methods are not usable, when the bilirubin concentration of serum is within 10mg/d L, the serum bilirubin concentration is 20mg/d L, when the serum bilirubin concentration is 30mg/d L, the perchloric acid and perchloric acid-glacial acetic acid methods are used, the interference deviation is within 8.81%, and the recovery rate is still between 90-110%, therefore, the perchloric acid-glacial acetic acid method is used, the recovery rate is in a better range and can be always between 90-110%, the jaundice serum is thoroughly treated, the limulus reagent reaction is not interfered, and the serum of a jaundice patient is preferably treated by the perchloric acid-glacial acetic acid method through comparison of the three methods.
Example 2
1. Lipemic sample:
the clinical concentration value of triglyceride in serum of healthy people is 1.98 mmol/L, the concentration of serum triglyceride in ordinary patients is about 2 times that of triglyceride in serum of healthy people, namely about 3.96 mmol/L, the concentration of serum triglyceride in ordinary patients is about 5-10 times that of triglyceride in serum of healthy people, namely about 9.9 mmol/L-19.8 mmol/L.
2.1 requirements of the experiment
All instruments in direct contact with the reagents should be sterile and free of pyrogen contamination.
1.2 Experimental methods
Three test samples (control, triglyceride addition and endotoxin recovery) were made per healthy population serum sample. Each test specimen was divided into four times.
1.3 samples
The control sample is prepared by mixing 0.9m L serum sample (No. 1-6) +0.1m L normal saline.
Interference sample 1A serum sample (No. 1-6) +0.1m L39.6.6 mmol/L triglyceride standard of 0.9m L healthy population was mixed and added at a concentration of 3.96 mmol/L.
Recovery of sample 1. mu. L interference sample + 10. mu. L0.5 EU/m L endotoxin solution → 100. mu. L serum with 0.05EU/m L endotoxin and 3.96 mmol/L triglyceride added.
Interference sample 2, 0.9m L serum sample from healthy population (No. 1-6) +0.1m L100 mg/d L triglyceride standard was mixed and added at 10mg/d L.
Recovery of sample 2. mu.1 interference sample + 10. mu. L0.5 EU/m L endotoxin solution → 100. mu. L serum with the addition of 0.05EU/m L endotoxin and 19.8 mmol/L triglyceride.
1.4 serum treatment method
The method comprises the following steps: heating dilution method
Control sample (No. 1-6), interference sample 1 (No. 1-6), recovered sample 1 (No. 1-6), interference sample 2 (No. 1-6), and recovered sample 2 (No. 1-6) were examined with water → 75 ℃ water bath for 10min → bacterial endotoxin test kit to determine endotoxin content, each 100. mu. L + 900. mu. L.
The method 2 comprises the following steps: perchloric acid process
Control sample (Nos. 1 to 6), interference sample 1 (Nos. 1 to 6), recovery sample 1 (Nos. 1 to 6), interference sample 2 (Nos. 1 to 6), and recovery sample 2 (Nos. 1 to 6) each 100. mu. L + 100. mu. L0.18.18N NaOH → 37 ℃ water bath for 5min → 100. mu. L0.32.32N HClO4Water bath at 37 ℃ for 10min → 200 μ L0.18N NaOH → 500 μ L0.2N Tris-HCl (pH 8.0) → 3000rpm centrifugation for 10min → determination of endotoxin content by bacterial endotoxin detection kit.
The method 3 comprises the following steps: perchloric acid-glacial acetic acid process
Control sample (Nos. 1 to 6), interference sample 1 (Nos. 1 to 6), recovery sample 1 (Nos. 1 to 6), interference sample 2 (Nos. 1 to 6), and recovery sample 2 (Nos. 1 to 6) each 100. mu. L + 120. mu. L0.2.2 mo L/L HClO4+80 μ L0.1 mo L/L glacial acetic acid → 37 ℃ water bath for 10min → 200 μ L0.25.25 mo L/L NaOH +500 μ L0.2 mo L/L phosphate buffer → bacterial endotoxin test kit to determine endotoxin content.
1.5 results (unit: EU/m L)
We summarize the following table based on the results of the interference and recovery tests:
(Note: clinical empirical data, serum triglyceride in lipemia is generally about 3-5 mmol/L).
From visual inspection, the serum is slightly turbid and whitened when the concentration of the common lipemia serum triglyceride is in the range of 1.98-3.96 mmol/L, at the moment, when the interference deviation is lower than-28% when measured by a thermodilution method and a perchloric acid method, the recovery rate is outside the range of 50-200%, neither method is available, when the concentration of the lipemia serum triglyceride is in the range of 1.98-3.96 mmol/L and the concentration of the triglyceride is 19.8 mmol/L, the interference deviation is within 7.72% by using the perchloric acid-glacial acetic acid method, and the recovery rate is still in the range of 90-110%.
Example 3
1 hemolyzed sample:
clinically, the hemoglobin value of the blood of healthy people is 120 g/L, the serum of healthy people does not contain hemoglobin, the hemoglobin value of slightly hemolytic serum is 1.2 g/L, the hemoglobin value of moderately hemolytic serum is 2.4 g/L, and the hemoglobin value of severely hemolytic serum is 4.8 g/L. from the visual inspection, the blood serum is reddish when the hemoglobin concentration of slightly hemolytic serum is within 1.2 g/L. the mathematical grade 2-fold addition method is used in the test, and the blood serum samples used for the test are 1 time, 2 time and 4 time of the hemoglobin concentration value of slightly hemolytic serum, namely, the concentration values are 1.2 g/L, 2.4 g/L and 4.8 g/L.
1.1 requirements of the experiment
All instruments in direct contact with the reagents should be sterile and free of pyrogen contamination
1.2 Experimental methods
Six healthy human serum samples were taken. Three test samples (control, added hemoglobin and endotoxin) were made for each healthy population serum sample. Four parallel tubes were tested per sample.
1.3 samples
The control sample is prepared by mixing 0.9m L serum sample (No. 1-6) +0.1m L normal saline.
Interference sample 1, 0.9m L serum sample of healthy population (No. 1-6) +0.1m L12 g/L hemoglobin standard is mixed, and the addition concentration is 1.2 g/L.
Recovery of sample 1. mu. L interference sample + 10. mu. L0.500 EU/m L endotoxin solution → 100. mu. L serum with 0.050EU/m L endotoxin and 1.2 g/L hemoglobin added.
Interference sample 2, 0.9m L serum sample of healthy population (No. 1-6) +0.1m L24 g/L hemoglobin standard was mixed and added at 2.4 g/L.
Sample 2 was recovered by taking 90 μ L perturbed sample 2+10 μ L0.500 EU/m L endotoxin solution → 100 μ L serum plus 0.050EU/m L endotoxin solution and 2.4 g/L hemoglobin.
Interference sample 3, 0.9m L serum sample of healthy population (No. 1-6) +0.1m L48 g/L hemoglobin standard was mixed and added at 4.8 g/L.
Sample 3 was recovered by taking 90 μ L perturbed sample 3+10 μ L0.500 EU/m L endotoxin solution → 100 μ L plus 0.050EU/m L endotoxin solution and 4.8 g/L hemoglobin in serum.
1.4 serum treatment method
The method comprises the following steps: heating dilution method
Control samples (No. 1-6), interference samples 1 (No. 1-6), recovery samples 1 (No. 1-6), interference samples 2 (No. 1-6), recovery samples 2 (No. 1-6), interference samples 3 (No. 1-6), and recovery samples 3 (No. 1-6) were examined by water → 75 ℃ water bath for 10min → determination of endotoxin content by bacterial endotoxin test kit, 100. mu. L + 900. mu. L, respectively.
The method 2 comprises the following steps: perchloric acid process
Control sample (Nos. 1 to 6), interference sample 1 (Nos. 1 to 6), recovery sample 1 (Nos. 1 to 6), interference sample 2 (Nos. 1 to 6), recovery sample 2 (Nos. 1 to 6), interference sample 3 (Nos. 1 to 6), and recovery sample 3 (No. 1 to 6) each 100. mu. L + 100. mu. L0.18.18N NaOH → 37 ℃ water bath 5min → 100. mu. L0.32N HClO4Water bath at 37 ℃ for 10min → 200 μ L0.18N NaOH → 500 μ L0.2N Tris-HCl (pH 8.0) → 3000rpm centrifugation for 10min → determination of endotoxin content by bacterial endotoxin detection kit.
The method 3 comprises the following steps: perchloric acid-glacial acetic acid process
Control sample (1-6), interference sample 1 (1-6), recovered sample 1 (1-6), interference sample 2 (1-6), recovered sample 2 (1-6), interference sample 3 (1-6), and recovered sample 3 (1-6) each 100. mu. L + 120. mu. L0.2.2 mo L/L HClO4+80 μ L0.1 mo L/L glacial acetic acid → 37 ℃ water bath for 10min → 200 μ L0.25.25 mo L/L NaOH +500 μ L0.2 mo L/L phosphate buffer → bacterial endotoxin test kit to determine endotoxin content.
1.5 results (unit: EU/m L)
We summarize the following table based on the results of the interference and recovery tests:
The conclusion is that when the concentration of hemoglobin in slightly hemolytic serum is within 0.8 g/L-1.2 g/L, the serum is light red, and at the moment, the interference deviation is up to more than 2000% by using a thermodilution method and perchloric acid to measure, the recovery rate is outside the interval of 50-200%, neither method can treat hemolytic serum, and when the concentration of hemoglobin in slightly hemolytic serum is within 0.8 g/L-1.2 g/L, and the concentration of hemoglobin in moderately hemolytic serum is 2.4 g/L, and the concentration of hemoglobin in serum is 4.8 g/L, the perchloric acid-glacial acetic acid method developed by the inventor is adopted, the interference deviation and the recovery rate are still within a good range, the interference deviation is less than 10%, and the recovery rate is within the interval of 90-110%.
By comparing the three methods, the hemolyzed serum is best treated by the perchloric acid-glacial acetic acid method.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. A method for treating icterus, hemolysis and lipemia with serum is characterized in that the treating liquid contains (1) 0.2mo L/L perchloric acid, 0.1mo L/L glacial acetic acid, (2) 0.25mo L/L NaOH, (3) 0.2mo L/L phosphate buffer;
the 0.2mo L/L phosphate buffer solution is 0.2 mol/L potassium dihydrogen phosphate solution and 0.2 mol/L sodium hydroxide solution;
adding 100 mu L serum into 120 mu L0.2 mo L0/L1 perchloric acid +80 mu L20.1.1 mo L/L glacial acetic acid, mixing uniformly, carrying out water bath at 37 ℃ for 10min, adding 200 mu L0.25 mo L/L NaOH and 500 mu L0.2 mo L/L phosphate buffer solution, and determining the endotoxin content by using a bacterial endotoxin detection kit.
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| US3677707A (en) * | 1968-10-28 | 1972-07-18 | Abbott Lab | Method of determining haemoglobin |
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| US3677707A (en) * | 1968-10-28 | 1972-07-18 | Abbott Lab | Method of determining haemoglobin |
| CN103226142A (en) * | 2012-01-30 | 2013-07-31 | 南京长澳医药科技有限公司 | Method for detecting leucogen concentration in blood plasma |
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| Monitoring Propofol Serum Levels by Rapid and Sensitive Reversed-Phase High-Performance Liquid Chromatography During Prolonged Sedation in ICU Patients;I. Pavan et al;《Journal of Chromatographic Science》;19921231;第30卷;第164-166页 * |
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