CN107991409B - Method for simultaneously measuring 12 sulfonamides in blood plasma by adopting high-efficiency synthetic phase chromatography - Google Patents
Method for simultaneously measuring 12 sulfonamides in blood plasma by adopting high-efficiency synthetic phase chromatography Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses a high Performance synthetic phase Chromatography (UPC) for simultaneously separating and determining 12 sulfonamides in blood plasma2) A method. Adding acetonitrile into a plasma sample, carrying out vortex mixing and centrifugation, filtering supernatant, and then adopting UPC2Method determination, using Acquity UPC2TMBEH chromatography column, CO2And methanol as a mobile phase, and performing gradient elution, wherein the temperature of a column incubator is 40 ℃, and the back pressure of the system is 2100 psi. The method realizes baseline separation of 12 sulfonamides within 6min, the lowest limit of quantitation is 0.5 mu g/mL, and the recovery rate is between 80.53 and 103.33 percent. The method is simple and accurate to operate, saves time, is more environment-friendly, and is suitable for simultaneously detecting various sulfonamides in blood plasma.
Description
Technical Field
The invention relates to a method for simultaneously measuring 12 sulfonamides in blood plasma by adopting high-efficiency synthetic phase chromatography.
Background
Sulfonamides (SAs) are a generic term for a class of drugs containing a sulfanilamide structure. The antibacterial agent mainly exerts an antibacterial effect by competitively combining with P-aminobenzoic acid (PABA) and influencing bacterial nucleoprotein synthesis, and has the advantages of wide antibacterial spectrum, low price, stable property and the like. Until now, clinical applications are still wide.
Early studies showed that the plasma concentrations of different patients were significantly different when the same dose of SAs was administered. The blood concentration is maintained at 50-100 mug/mL in the conventional experience treatment process. In the blood concentration range, the antibacterial effect of the drug is obvious, and in the face of severe infection, the proper increase of the blood concentration of the drug is proper, and the blood concentration of a patient is usually maintained at 120-. In order to fully study the pharmacodynamics and pharmacokinetics of SAs, it is necessary to establish a rapid, simple, high-throughput method for determining SAs in plasma.
Currently, many researchers have established a series of analysis methods for SAs in biological samples [ Yu H, Tao Y, Chen D, et al.J. Chromator B analytical technique Biomed Life Sci,2011,879(25):2653-2662.ZHao XT, Lin QB, Song H, et al.J. aggregate Food Chem,2011,59(18):9800-9805.Zhang S, Hu Q, Sun J, et al.Chin.legacy Patent Med.,2015,37(3): 542-. At home and abroad, SAs are more analyzed in biological samples, wherein HPLC (UHPLC) detection technology is most commonly used [ GaoR, Zhang J, He X, et al, anal Bioanal Chem.2010,398(1):451-461.Shaaban H, G Lo reckit. J Sep Sci,2012,35(2): 216) J Sep Sci ], SAs have similar structures, and the separation efficiency is to be improved no matter C18 columns or amino columns are adopted. Ultra-high performance integrated chromatography (UPC2) is a separation technology newly introduced by Waters company in the 8 th month of 2012, and the technology adopts the basic principle of supercritical fluid, so that the separation efficiency is high, and the technology has the advantages of being green, environment-friendly, economic and the like.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring 12 sulfonamides in blood plasma.
The method for simultaneously measuring 12 sulfonamides in blood plasma provided by the invention adopts high performance synthetic phase chromatography for measurement, and comprises the following steps:
1) pretreating a plasma sample to be detected:
taking a proper amount of a plasma sample to be detected, adding acetonitrile with the same volume, uniformly mixing, centrifuging, taking supernate, filtering the supernate with a 0.22 mu m filter membrane, and collecting filtrate to be detected;
2) the determination is carried out by adopting high performance phase-combination chromatography:
the high-efficiency combined phase chromatography adopts the following chromatographic conditions:
using Acquity UPC2A BEH chromatography column;
column temperature: 35-45 ℃, system back pressure: 1800psi-2700 psi;
mobile phase: supercritical CO2And methanol;
and (3) an elution mode: gradient elution;
the gradient elution procedure was:
0-5 min: supercritical CO2From 95% to 85%,
5.0-5.5 min: supercritical CO2From 85% to 80%,
5.5-6.0 min: supercritical CO2From 80% to 95%,
6.0-7.0 min: supercritical CO2The volume fraction of (A) is maintained constant at 95%;
the column flow rate is 1.55-1.65mL/min (preferably 1.6 mL/min);
the detector adopted by the high-efficiency phase-combination chromatography is a PDA (personal digital assistant) detector (diode array detector);
wherein the 12 sulfonamides are selected from the following substances: sulfamethoxazole (SMZ), Sulfamethoxazole (SMM), Sulfadimethoxypyrimidine (SMM), Sulfacetamide (STD), and sulfamethoxazolePyrimidine (SMR), Sulfadiazine (SDZ), Sulfathiazole (ST), Sulfapyridine (SPD), N4-Acetylsulfamethoxazole (N)4-Ac SMZ)、N4-acetylsulfanylmethyl pyrimidine (N)4-Ac SMR)、N4-acetylsulfadiazine (N)4-Ac SDZ)、N4-sulfacetamide thiazole (N)4-AcST)、N4-acetylsulfapyridine (N)4-Ac SPD)。
In the step 1), the uniform mixing adopts a vortex mode, and the vortex time is 0.5-1 min; the centrifugation condition is 6000-10000 rpm centrifugation for 6-12 min at 18-24 ℃.
In step 2), the Acquity UPC2The specification of the BEH chromatographic column is 100mm multiplied by 3mm, and the grain diameter of the filler is 1.7 mu m; the column temperature is: 40 ℃; the system back pressure is as follows: 2100 psi;
in the chromatographic condition, the sample temperature is 25 ℃, and the sample injection volume is 2 mu L.
The detection conditions of the PDA detector in the step 2) are as follows:
the 3D scanning range 210-.
The invention also aims to provide a method for simultaneously measuring the content of 12 sulfonamides in blood plasma by adopting high performance synthetic phase chromatography, which comprises the following steps:
1) preparation of the Standard Curve
Adding a series of mixed standard solutions of the 12 sulfonamides in concentration into a blank plasma sample, preparing according to the method for pretreating the plasma sample, detecting the obtained supernatant according to the high performance gel chromatography, and respectively recording the corresponding peak areas of the 12 sulfonamides at each concentration,
taking the peak area corresponding to each sulfonamide as the ordinate, taking the mass concentration of the corresponding sulfonamide as the abscissa, and performing linear weight regression to respectively prepare regression equations of the 12 sulfonamides;
2) determining the content of the 12 sulfonamides in the plasma sample to be tested
Preparing a blood plasma sample to be detected according to the sample preparation method, detecting the obtained supernatant according to the high performance gel chromatography, and respectively recording peak areas corresponding to the 12 sulfonamides; and respectively substituting the peak area value of each drug into a regression equation corresponding to the drug, and calculating to obtain the concentrations of the 12 sulfonamides in the blood plasma sample to be detected.
The method can be successfully used for the pharmacokinetic research of sulfonamides.
Until now, liquid chromatography (or tandem mass spectrometry) is still adopted in the conventional method for measuring SAs, but the method has long separation time, needs to improve the separation efficiency, has high consumption of organic reagents and needs to improve the environmental friendliness. In view of the above, the invention establishes a novel rapid, efficient and environment-friendly separation method for analyzing and determining SAs in biological samples. The established method has the following advantages: (1) the separation time is short, and the separation of 12 SAs can be realized within 6 minutes; (2) the separation efficiency is high, and all substances can achieve baseline separation; (3) clean and environment-friendly, and effectively reduces the use amount of organic reagents.
Drawings
FIG. 1 is a chromatogram of 12 sulfonamides (A): blank plasma; (B) the method comprises the following steps Blank plasma +12 sulfonamides.
Detailed Description
The method of the present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples 1,
1 materials and methods
1.1 instruments and reagents
Acquity UPC2(Waters corporation, USA) equipped with a Waters PDA detector; chromatography columns (all available from Waters, Milford, MA): (1) waters ACQUITY UPC2BEH(1.7μm,3×100mm I.D.),(2)WatersAcquity UPC2BEH 2-EP(1.7μm,3×100mm I.D.),(3)Acquity UPC2CSHFluoro-Phenyl(1.7μm,2.1×100mm I.D.)and(4)Acquity UPC2HSS C18SB (1.8 μm,3 × 100mm i.d.). Toledol model 204 electronic balance (METTLER, switzerland). PURELA Ultra MK2 model ultrapure water meter was purchased from ELGA, UK.
SAs control: sulfamethoxazole (SMZ, > 99%, w/w), sulfadimethoximidine (SMM, > 99%, w/w), Sulfacetamide (STD, > 99%, w/w), Sulfadiazine (SMR, > 99%, w/w), Sulfadiazine (SDZ, > 99%, w/w), Sulfathiazole (Sulfadazole, ST, > 99%, w/w), Sulfapyridine (Sulfapyridine, > 99%, w/w), all available from SPD. N is a radical of4-Acetylsulfamethoxazole (N)4-Ac SMZ,>95%,w/w)、N4-acetylsulfanylmethyl pyrimidine (N)4-Ac SMR,>95%,w/w)、N4-acetylsulfadiazine (N)4-Ac SDZ,>95%,w/w)、N4-sulfacetamide thiazole (N)4-Ac ST,>95%,w/w)、N4-acetylsulfapyridine (N)4-Ac SPD, > 95%, w/w), all available from Toronto Research Chemicals, Canada. Methanol, ethanol and isopropanol (Merck, Germany, chromatographically pure), ammonium formate and ammonium acetate (Agilent, chromatographically pure, USA). CO 22(food grade) purchased from Beijing Lvyoxon Tiangang technology Ltd, and the laboratory water was ultrapure water.
1.2 preparation of Standard solution
Accurately weighing 12 sulfanilamide and metabolite reference substances 0.01g (accurate to 0.0001g), dissolving with methanol, and metering to 100mL volumetric flask to obtain 100 μ g/mL mixed standard stock solutions. Freezing and storing at-20 deg.C in dark, diluting with methanol to obtain standard working solution with different mass concentration, and storing at 4 deg.C in dark.
1.3 chromatographic conditions
Chromatographic conditions are as follows: using Acquity UPC2BEH column (100 mm. times.3 mm,1.7 μm), column temperature: 40 ℃, system back pressure: 2100psi, sample temperature 25 ℃ and sample volume 2. mu.L. The mobile phases are methanol (phase B) and supercritical CO respectively2(phase A), column flow rate 1.6mL/min, mobile phase gradient elution program is shown in Table 1.
TABLE 1 gradient elution procedure
1.4PDA Condition
The 3D scanning range 210-.
1.5 sample pretreatment
Precisely measuring 0.5mL of plasma sample, placing in a 2mL centrifuge tube, adding 0.5mL of acetonitrile, vortex and mixing uniformly for 30s, centrifuging at 20 ℃ at 10000rpm for 10min, taking supernatant, filtering with a 0.22 μm filter membrane, and detecting on a computer.
2 results
2.1 specificity
Blank plasma is taken, a sample solution is prepared according to the item of '1.5 sample pretreatment', sample introduction is carried out according to the '1.3 chromatographic condition', a blank plasma chromatogram has no endogenous impurity peak within 2-6min, SAs determination is not influenced, and the method has high specificity. The chromatogram is shown in FIG. 1.
2.2 Standard Curve and Linear Range
Preparing 2, 4, 10, 20, 40 and 100 mu g/mL mixed standard solutions, precisely measuring 0.5mL of the reference substance stock solutions with the mass concentrations, respectively placing the reference substance stock solutions into 2mL centrifuge tubes, adding 0.5mL of blank plasma, uniformly mixing to obtain 1, 2, 5, 10, 20 and 50 mu g/mL series sample plasma, treating according to the method under the item of 1.5 sample pretreatment, and sampling 2 mu L of sample. Calculating to obtain the detection Limit (LOD) of the method according to the S/N which is more than or equal to 3, calculating to obtain the quantitative Limit (LOQ) of the method according to the S/N which is more than or equal to 10, and performing linear weight regression by taking the peak area of each sulfanilamide as a vertical coordinate and the mass concentration c (mu g/mL) of each sulfanilamide substance as a horizontal coordinate to obtain a regression equation. See table 2 for details.
TABLE 212 regression equation, Linear Range and lower quantitative limits for sulfonamide drugs
2.3 precision and accuracy
Taking blank plasma, preparing 6 parts of each of 3 plasma samples (5, 50 and 100 mu g/mL) with low, medium and high mass concentrations according to the item of 'standard curve and linear range', operating according to the method under the item of '1.5 sample pretreatment', carrying out batch measurement with the standard curve, repeatedly measuring for 4d, and obtaining the precision (RSD) of the method by analyzing variance of the measurement result. The result indicates that the method has the daily precision RSD of 1.5-4.1%, the daytime precision RSD of 1.3-5.2% and the accuracy of 95-99%, and all meet the measurement requirement of biological samples.
2.4 stability test
Taking 0.5mL of blank plasma, placing the blank plasma in a 2mL sample tube, respectively and precisely adding 0.5mL of 10, 80 and 160 mu g/mL of sulfanilamide reference substance stock solutions, uniformly mixing to prepare 5, 40 and 80 mu g/mL of series plasma samples with low, medium and high 3 mass concentrations, treating according to the method under the item of 1.5 sample pretreatment, and taking 2 mu L of sample injection. And preparing 6 parts of each of 3 samples with mass concentration by the same method, standing 2 parts of the samples at room temperature for 5 hours, repeatedly freezing and thawing 2 parts of the samples for 5 times, storing 2 parts of the samples for 20 days under the freezing condition, treating the samples by the same method, and sampling 2 mu L of supernate. The mass concentration of each sample was determined from the calibration curve and compared with the sample of the corresponding mass concentration of 0 h. The results show that: the plasma sample is placed for 5h at room temperature, frozen and thawed for 5 times and stored for 20d at the temperature of minus 20 ℃ and has good stability.
2.5 extraction recovery
Taking 0.5mL of blank plasma, placing the blank plasma in a 2mL sample tube, respectively and precisely adding 0.5mL of sulfonamide reference substance stock solutions of 10, 80 and 160 mu g/mL, uniformly mixing to prepare plasma samples with low, medium and high mass concentrations of 5, 40 and 80 mu g/mL, processing the samples according to the item of '1.5 sample pretreatment', respectively taking 2 mu L of each solution for sample injection, and continuously measuring for 5 times. The recovery yields (n ═ 5) for the low, medium and high 3 mass concentrations of different SAs are shown in table 3.
TABLE 3 recovery rate of extraction
Until now, liquid chromatography (or tandem mass spectrometry) is still adopted in the conventional method for measuring SAs, but the method has long separation time, needs to improve the separation efficiency, has high consumption of organic reagents and needs to improve the environmental friendliness. In view of this, the present research establishes a novel rapid, efficient and environmentally friendly separation method, which is intended for the analytical determination of the SAs in biological samples. The established method has the following advantages: (1) the separation time is short, and the separation of 12 SAs can be realized within 6 minutes; (2) the separation efficiency is high, and all substances can achieve baseline separation; (3) clean and environment-friendly, and effectively reduces the use amount of organic reagents. In the research process of the method, the experiment mainly inspects the influence of different separation factors on the separation effect, and the method comprises the following specific steps:
3.1 influence of the chromatography column on the separation effect of different SAs:
the 12 SAs in the experiment have the characteristics of similar structure height and difficult separation. The results of experiments on the BEH, BEH2-EP, CSH Fluoro-Phenyl and HSS C18SB chromatographic columns respectively show that under the same experimental conditions, the BEH chromatographic column obtains the best separation effect (the base line separation of each drug pair is achieved, R is more than 1.5), and the rest chromatographic columns can not achieve the base line separation of all substances. The elution sequence was: (1) SMZ, (2) SMM, (3) N4-Ac SMZ,(4)STD,(5)SMR,(6)SPD,(7)SDZ,(8)N4-Ac SMR,(9)N4-Ac SDZ,(10)N4-Ac SPD,(11)ST,(12)N4-Ac ST (see FIG. 1).
3.2 Effect of modifiers on the separation Effect of different SAs:
UPC2the most common mobile phase for the system is CO2Meanwhile, a small amount of organic reagent is added as a modifier to promote separation, different types of modifiers have different influences on the separation of the target object, and the separation effect of methanol, ethanol and acetonitrile as the modifiers is respectively investigated in the experiment. Selection of 12 SAs with 3 reagentsThe selectivity is also greatly different, wherein the methanol separation time is shortest and the separation effect is optimal, the separation degree of all compounds can reach more than 1.5, and when ethanol and acetonitrile are used as mobile phases, the separation degree of partial sulfanilamide is required to be improved and the separation time is longer.
3.3 influence of chromatographic column temperature and system backpressure on separation effect of different SAs:
in UPC2In the system development process, the chromatographic column temperature and the system backpressure are two important parameters for separation optimization, and from the separation mechanism, the supercritical CO can be obviously changed by changing the backpressure value or the chromatographic column temperature2And thus its elution capacity and selectivity. The effect of column temperature (30, 35, 40, 45, 50, 55 ℃) and system pressure (1500, 1800, 2100, 2400, 2700, 3000psi) on analyte separation was examined in this experiment.
The time of the compound peak-out gradually increases with the temperature increase from 30 c, and it is considered that the temperature increase lowers the density of the supercritical fluid and lowers the elution ability of the mobile phase, resulting in an increase in the retention time of the compound. Compounds SMM and N below 35 ℃4the-Ac SMZ failed to separate at baseline, and the degree of separation of the two compounds increased with increasing temperature. At a temperature of over 45 ℃, N4The Ac SMZ and STD failed to separate from baseline, and the degree of separation decreased with increasing temperature.
Starting at 1500psi, the time to peak compound gradually decreased as the backpressure increased. This is thought to be because an increase in back pressure increases the density of the supercritical fluid, increases the elution capacity of the mobile phase, and decreases the retention time of the compound. Backpressure values of 1800psi and below, SMM and N4Ac SMZ failed to separate baseline and the separation of the two compounds tended to increase with increasing backpressure; 2700psi and above, N4the-Ac SMZ and STD failed to separate from baseline and the separation of the two compounds tended to decrease with increasing backpressure.
Considering the influence of the temperature and the system back pressure on the separation degree, the optimal conditions of the column temperature of 40 ℃ and the system back pressure of 2100psi are selected.
3.4 Effect of elution procedure on separation Effect of different SAs:
this study examined 80%, 85%, 90%, 95% CO respectively2Isocratic elution under four conditions, and 80% CO is found2Isocratic elution causes all chromatographic peaks to elute within 1.8 minutes, baseline separation cannot be achieved, 85% CO2All substances eluted at equal degree will peak within 3.2min, the separation effect is not satisfactory, and 90% CO2Isocratic elution, all substances peaked within 9min, but p-sulfamonomethoxine and N4The undissociated-acesulfame-methyloxazole, 95% CO2Isocratic elution, the separation time is more than 10min, the peak shape is flat and wide and cannot be satisfied, and gradient elution is adopted to synthesize the above results.
5 gradient programs were examined separately in the experiment, and finally the elution program was found: 0 min: 95% CO2;5min:85%CO2;5.5min:80%CO2;6min:95%CO2;7min:95%CO2A baseline separation (resolution > 1.5) can be obtained for all test substances.
3.5 Effect of chromatographic flow Rate on separation of different SAs:
the effect of flow rates of 1.2-2.2mL/min on separation was investigated separately in this study. The results show that the compound peak time is significantly reduced with increasing flow rate. Considering the comprehensive separation effect and the separation rate, the optimal effect is achieved by selecting the flow rate of 1.6 mL/min.
Claims (9)
1. A method for simultaneously measuring 12 sulfonamides in blood plasma adopts high performance synthesis chromatography for measurement, and comprises the following steps:
1) pretreating a plasma sample to be detected:
taking a proper amount of a plasma sample to be detected, adding acetonitrile with the same volume, uniformly mixing, centrifuging, taking supernate, filtering the supernate with a 0.22 mu m filter membrane, and collecting filtrate to be detected;
2) the determination is carried out by adopting high performance phase-combination chromatography:
the high-efficiency combined phase chromatography adopts the following chromatographic conditions:
using Acquity UPC2A BEH chromatography column;
column temperature: 35-45 ℃, system back pressure: 1800psi-2700 psi;
mobile phase: supercritical CO2And methanol;
and (3) an elution mode: gradient elution;
the gradient elution procedure was:
0-5 min: supercritical CO2From 95% to 85%,
5.0-5.5 min: supercritical CO2From 85% to 80%,
5.5-6.0 min: supercritical CO2From 80% to 95%,
6.0-7.0 min: supercritical CO2The volume fraction of (A) is maintained constant at 95%;
the flow rate of the column is 1.55-1.65 mL/min;
the detector adopted by the high-efficiency phase-combination chromatography is a PDA detector;
wherein the 12 sulfonamides are selected from the following substances: sulfamethoxazole, sulfamonomethoxine, sulfacetamide, sulfamethazine, sulfadiazine, sulfathiazole, sulfapyridine, N4-acetylsulfamethoxazole, N4-acetylsulfanylmethyl pyrimidine, N4-acetylsulfadiazine, N4-sulfacetamide thiazole, N4-sulfacetamide pyridine.
2. The method of claim 1, wherein: in the step 1), the uniform mixing adopts a vortex mode, and the vortex time is 0.5-1 min; the centrifugation condition is 6000-10000 rpm centrifugation for 6-12 min at 18-24 ℃.
3. The method according to claim 1 or 2, characterized in that: in the step 2), the Acquity UPC2The specification of the BEH chromatographic column is 100mm multiplied by 3mm, and the grain diameter of the filler is 1.7 mu m; the column temperature is: 40 ℃; the system back pressure is as follows: 2100 psi;
in the chromatographic condition, the sample temperature is 25 ℃, and the sample injection volume is 2 mu L.
4. The method of claim 1, wherein: in the step 2), the detection conditions of the PDA detector are as follows:
the 3D scanning range 210-.
5. A method for simultaneously determining the content of 12 sulfonamides in blood plasma adopts high performance synthesis chromatography for determination, and comprises the following steps:
1) preparation of the Standard Curve
Adding a series of concentrations of the mixed standard solution of 12 sulfonamides in the claim 1 into a blank plasma sample, processing the plasma sample according to the method of the step 1) in the claim 1, detecting the obtained supernatant according to the high performance gel chromatography in the claim 1, and respectively recording the peak area corresponding to each concentration of the 12 sulfonamides,
taking the peak area corresponding to each sulfonamide as the ordinate, taking the mass concentration of the corresponding sulfonamide as the abscissa, and performing linear weight regression to respectively prepare regression equations of the 12 sulfonamides;
2) determining the content of the 12 sulfonamides in the plasma sample to be tested
Treating a plasma sample to be detected according to the method in the step 1) in the claim 1, detecting the obtained supernatant according to the high performance chromatography in the claim 1, and respectively recording peak areas corresponding to the 12 sulfonamides; substituting the peak area value of each drug into the regression equation corresponding to the drug prepared in the step 1), and respectively calculating the concentrations of the 12 sulfonamides in the blood plasma sample to be detected.
6. The method of claim 5, wherein: in the step 1), the uniform mixing adopts a vortex mode, and the vortex time is 0.5-1 min; the centrifugation condition is 6000-10000 rpm centrifugation for 6-12 min at 18-24 ℃.
7. The method according to claim 5 or 6, characterized in that: in the step 2), the Acquity UPC2The specification of the BEH chromatographic column is 100mm multiplied by 3mm, and the grain diameter of the filler is 1.7 mu m; the column temperature is: 40 ℃; the system back pressure is as follows: 2100 psi;
in the chromatographic condition, the sample temperature is 25 ℃, and the sample injection volume is 2 mu L.
8. The method of claim 5, wherein: in the step 2), the detection conditions of the PDA detector are as follows:
the 3D scanning range 210-.
9. Use of the method of any one of claims 1 to 8 for studying the pharmacokinetics of sulfonamides; the sulfanilamide drug is selected from at least one of the following drugs: sulfamethoxazole, sulfamonomethoxine, sulfacetamide, sulfamethazine, sulfadiazine, sulfathiazole, sulfapyridine, N4-acetylsulfamethoxazole, N4-acetylsulfanylmethyl pyrimidine, N4-acetylsulfadiazine, N4-sulfacetamide thiazole, N4-sulfacetamide pyridine.
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