ANALYTICAL CHEMISTRY ARTICLE SUBJECT STUDYING THE ANALYTICAL CONDITIONS OF SULFONAMIDES BY CHROMATOGRAPHIC METHOD.

Socialist Republic of Vietnam Independence-Freedom-Happiness SCHOOL OF MEDICINE VIET NAM NATIONAL UNIVERSITY HO CHI MINH CITY ANALYTICAL CHEMISTRY ARTICLE SUBJECT STUDYING THE ANALYTICAL CONDITIONS OF SULFONAMIDES BY CHROMATOGRAPHIC METHOD CLASS: D3-2020 GROUP MEMBERS Mai Nguyễn Đăng 207720201006 Nguyễn Xuân Hiệp 207720201014 Phạm Nhật Hoàng 207720201015 Nguyễn Thanh Huyền 207720201017 Nguyễn Tấn Tài 207720201033 Binh Duong, September 29, 1| P a g e 1| P a g e Abstract HPLC optimization includes choosing the detector's wavelength, the stationary phase, and the mobile phase's pH, composition, and speed The separation conditions are also optimized Linear interval survey; detection and quantification limits; Analyze the measurement's accuracy and repeatability fecal sample handling conditions optimization Choose a representative treatment approach, then assess the effectiveness of recovery To examine various shrimp samples, develop analytical processes and use research processes Keyword Chromatographic method;  Analytical chemistry;  Sulphonamides;  Antibiotics;  Shrimp; Identify problem: Shrimp farming is now the most significant industry in Vietnam, and it is also the aquaculture development plan's primary goal The fast growth of shrimp aquaculture contributes to a rise in medication and chemical usage MTD, a member of the broad range antibacterial medication family SAs, is frequently used in shrimp farming to prevent and cure a variety of bacterial illnesses in animals If the animals are not given time to clean themselves after using these chemicals, there will be leftover antibacterial compounds that might be harmful to health Metronidazole sulfaguanidin (SGU), sulfamethoxypyridazine (SMP), sulfadoxine (SDO), and sulfamethoxazole (SMX), all of which are members of the SAs antibacterial family and are often employed in blankets, are the analytical topics that we decided to research Based on this need, we chose the HPLC reverse phase adsorption method as the research method, with a UV-Vis detector as the linked detector 1.1 Introduction to Sulphonamides (SAs) and Metronidazole (MTD) 1.1.1 Molecular structure: Figure 1: General molecular structure of SAs When we replace groups R1, R2 with different radicals, we have different SAs Figure 2: Structure of Metronidazole (MTD) 2| P a g e It is a nitroimidazole-family antibiotic used mostly to treat anaerobic bacteria and protozoa MTD is a component of animal feed, also an antibiotic used in aquaculture (with the trade name Enro DC) 1.1.2 Physical and chemical properties of the Sulphonamides, Metronidazole 1.1.2.1 Physical properties: SAs are white or pale yellow crystals, odorless, sparingly soluble in water, soluble in acids and alkaline solutions (except sulfaguanidine).  MTD is a crystalline or crystalline powder that is odorless, yellowish, stable in the air, and gradually darkens when exposed to light The melting point ranges between 159 and 163℃ Metronidazole hardly dissolves in acetone and water 1.1.2.2 Chemical properties:  SAs are amphoteric SAs form complex salts that precipitate with Ag+ ions, and precipitate colored complexes with Cu2+, Co2+ ions, In the primary amine group of SAs, there are free electron pairs, helping SAs to carry out charge transfer complexation with phenosafranine (PSF) giving a purple complex with a maximum absorption wavelength at 270-273 nm 1.1.3 Pharmacological properties and spectrum of action of Sulphonamides, Metronidazole: With therapeutic doses, SAs not kill bacteria, only make bacteria weaker, unable to grow and reproduce, easily destroyed by white blood cells SAs is a broad-spectrum antibiotic: they are effective against many anthrax bacteria, cholera bacteria, Shigella, E.coli, and bacilli Along with protozoa and anaerobes, Metronidazole also has a wide range of action against Eubacterium, Peptococcus, Peptostreptococcus, Fusobacterium Veillonella, Clostridium, including C.difficile and C.perfringens It works well against isolates of B fragilis that are clindamycin resistant 1.1.4 Mechanism of antibacterial action of Sulphonamides, Metronidazole 1.1.4.1 Antibacterial mechanism of SAs: Inhibits folic acid metabolism Inhibits the synthesis of folic acid by bacteria 1.1.4.2 Antibacterial mechanism of Metronidazole: Metronidazole's action against obligate anaerobes occurs through a four-step process: Attack on microorganisms Decreased activation by intracellular protein transport Decreased cell-mediated subunit interactions - toxic intermediates interact with host DNA, leading to DNA strand breakage and DNA strand destruction Breakdown of cytotoxic intermediates - toxic intermediates break down into inactive end products 3| P a g e Design the experimental procedure  2.1 Research method In the article, samples of prawn are collected and examined, with highly complicated components Therefore, high performance liquid chromatography (HPLC) was chosen to survey the conditions of extraction and quantitative analysis 2.1.1 General principles and equipment required for HPLC High performance liquid chromatography (HPLC) is a segregation of compounds based on the movement of solutes through a separation column that has either pellicular and porous material as the inner coating Solute will move through the mobile phase at different velocities, depending on its distribution constant 2.1.2 Quantitative analysis using HPLC In the chosen conditions, every substance has a unique quantity t Ri, indicating the amount of time that a certain substance is moving through the column We can use this time to conduct qualitative analysis onto the substance based on a standard sample Then, quantify the substance based on analytical properties of the result Normally, the concentration of substance is usually depending on the height (H) or area (S) of the peek     H = k.Cb   or   S = k.Cb     In which H: height of the peek S: area of the peek k: experimental constant (depends on the conditions of the experiment) b: nature constant, satisfied < b ≤ 2.2 Solid phase extraction When the amount of analyte is small, analyte enrichment through solid phase extraction is essential Moreover, food samples have a complicated composition of substance, with many types of fat beside the analytes Therefore, solid phase extraction is necessary to isolate the analyte quantitatively, remove and evict any impurities Conduct an experiment  3.1 Investigation of chromatographic conditions 3.1.1 Determination the wavelength of the detector Based on the similarity of the maximum absorption capacity, we decided to choose a common wavelength of 270 nm for substances MTD is more sensitive (320 nm); therefore, the detector has two channels of 320 nm and 270 nm for efficient quantification 4| P a g e Fig The absorption capacity of wavelength of substances 3.1.2 Exploring the separation ability of sulfonamides on RP-C18 column Retention time is calculated when the sample is loaded into the chromatographic separation column until the solute is eluted from the column at the point of maximum concentration Retention time can determine the order of a substance's appearance.  The retention time of standard solution 1,0ppm on chromatographic mobile phase (20% ACN – 80% buffer solution acetate, buffer solution concentration 10mM, pH=4,5) Temp: 30oC Speed of mobile: 1ml/min Detector UV-Vis: channels 270 nm and 320 nm No Retention time (min) Analytes 3,30 SGU 4,48 MTD 8,49 SMP 13,23 SDO 15,08 SMX Table 1: Retention time and peak order of analytes Purpose: realizing the specific retention time of each substance as a basis for qualitative and quantitative determination of sulphonamides, metronidazole in research subjects 3.2 Determination stationary phase To research separation ability and determine SAs, MTD which are polarizers Therefore, most published works use separation columns containing reversed phase stuffing such as RP- C4, RP - C12 RP - C18 column is chosen to separate above substances with packed particle size of µm.  3.3 Mobile phase optimization 3.3.1 Acetate buffer concentration of mobile phase The selected buffer concentration values are in the range of - 20mM As a result, the capacity factor is almost unchanged Moreover, the buffer concentration did not have a significant effect on the separation ability, peek appearance time and peek chromatographic area The capacity factor, the separation ability, peek appearance time and peek chromatographic area are almost not changed show that the reflecting separation ineffectively At a certain buffer concentration in the mobile phase, the capacity factors of the analytes are significantly different, so it reflects effective separation between substances in the chromatographic process With the buffer concentration range, they show chromatographic 5| P a g e peek that are very sharp and distinct Based on these observations, the appropriate buffer concentration in the mobile phase was selected as 10mM Fig The capacity factors of analytes depend on the acetate buffer concentration 3.3.2 pH of buffer acetate Analytes are amphoteric with pKa values between 5.4 and 7.4 so the use of acidic buffer often gives sharp chromatographic peaks For that reason, we investigated the composition of buffer solution CH3COOH/CH3COONa with pKa=4.75 in the concentration range of 10mM with pH values: 3.5; 4.0; 4.5; 5.0; 5.5.  The higher the pH, the weaker the separation between SMX and SDO (pH=5.5), the lower pH, the long elution time (pH=3.5) At pH = 4.5, the chromatographic peaks have more distinct separation, more balanced peaks and less time for chromatographic peaks to appear In addition, there is a significant difference in volume coefficient After that, we chose a buffer solution having suitable pH Fig The capacity factors of analytes depend on the pH of buffer acetate 3.3.3 Ratio of mobile phase components The ratio of solvent components to the mobile phase affects the elution of the samples from the separation column As the mobile phase composition ratio changes, the elution force of the mobile phase changes As a result, it makes the change of the retention time and the volume coefficient of the analyte Buffer solution acetate has pH=4,5 and organic solution Acetonitrile (ACN) that change ratio of mobile phase components following below conditions: If we follow the way that ratio of mobile phase components is higher, the elution time from the column is faster Moreover, the volume coefficients are closer together and the 6| P a g e chromatographic peaks are not clearly separated that affect the area of the chromatographic peaks (like 30% ACN – 70% buffer) If we reduce the ACN rate, it takes a long time to elute the analyte In conclusion, experiment need an effective ratio of elution time (not too fast and not too slow), clear peek and chosen ratio is 20% ACN – 80% buffer Fig The capacity factors of analytes depend on ratio of mobile phase components 3.3.4 Speed of mobile phase The speed of the mobile phase is a decisive factor in the elution of substances in the chromatographic column, because it affects the process of establishing a solute equilibrium between the stationary and mobile phases The mobile phase speed is too small will cause the phenomenon of peak dispersion and increase the elution time but too large mobile phase speed lead to overlapping peaks Therefore, it is necessary to choose an appropriate mobile phase rate For a definite split column which has an optimal rate In this case, we survey mobile phase rate in the range from 0.6 to 1.4 ml/min with optimal conditions and conduct the above survey (acetate buffer with pH=4.5; buffer concentration 10mM; composition mobile phase consisting of 20% ACN-80% buffer), the results are obtained:  At the speed of 1ml/min, the separation is quite good and does not take much time to separate the substance from the column According to the figure, we choose a mobile phase rate of 1ml/min for the next experiments Fig The capacity factors of analytes depend on speed of mobile phase 7| P a g e Analyze the experimental data 4.1 Survey to establish a standard curve in the concentration range of 0.05 1,000ppm From the conditions that have been optimized above, proceed to observe the linear interval of the measurement with the following condition Stationary phase RP C18 (25cmì4.6cm; 5àm Mobile phase 20% ACN-80% acetate buffer 10mM (pH=4.5) Mobile phase rate ml/min Separation column temperature 30oC Detector UV: 270 nm, 320nm Concentration range 0.05ppm – 1.00 ppm Quantitative method Charge of peak Table Optimal conditions for chromatography 8| P a g e Fig.8 Standard curve of analyte in the concentration range 0.05 – 1.00ppm a Sulfaguanidine Standard Curve (SGU) b Metronidazole (MTD) Standard Curve c Standard curve of Sulfamethoxypyridazine (SMP) d Standard curve of Sulfadoxine (SDO) e Standard curve of Sulfamethoxazole (SMX) 4.2 Limit of Detection (LOD); Limit of Quantification (LOQ) Standard deviation: s= √ ∑ ( Si−Stb )2 n−1 In there:   Si - Value of the analyzed signal at the i-time measurement (mAU.s) Stb - Average signal value of i measurements (mAU.s) LOD (ppm):   LOD = LOQ (ppm): LOQ = Sy B 10 Sy B In there:   Sy - The standard deviation of the blank, also determined by the regression equation B - The slope in the regression equation Substance Coefficient of angle b Standard deviation LOD (ppm) LOQ (ppm) SGU 117814,44 772,12 0,020 0,066 MTD 124010,14 1028,82 0,025 0,083 SMP 129146,83 1017,35 0,024 0,079 SDO 139792,32 1336,46 0,029 0,096 SMX 126666,70 502,18 0,012 0,040 9| P a g e Table LOD, LOQ calculated by regression equation 4.3 Measurement accuracy, repeatability: To evaluate measurement error, select analytical samples whose concentrations are in the linear range at the beginning, middle, and end of the linear range investigated We proceed to prepare three standard samples with a concentration of 0.08; 0.4 and 0.8ppm with the same condition as the linear interval survey condition Through the analysis table and the above calculation results, we see that for samples with high concentration, the error is small, for samples with small concentration, the error is large According to statistical theory, the allowable error is within 15%, so for the concentration range investigated, the accuracy of this measurement is reliable Propose a solution:  CHƯƠNG 5.1 The optimal conditions for chromatography have been selected: RP-C18 column: 25 cm × 4.6 mm; µm UV-VIS detector:  two-channel = (1) 270 nm (2) 320 nm, Rise time = 0.1 s; Range = 0.01 AUFS Recorder: paper speed = mm/min; write potential = 10 mV Mobile phase composition: acetate buffer (pH = 4.5) 10mM /aceto-nitrile: 80/20 (V/V) Mobile phase speed: ml/min 5.2 The analytical method has been evaluated: The linear range of sulfates: 0.05 – 1.00ppm LOD: 0.012 – 0.029 ppm LOQ: 0.040 - 0.096 ppm Coefficient of variation: 0.2% - 5% in the concentration range 0.08 - 0.8ppm 5.3 Survey of real samples: Select the appropriate sample handling procedure, the recovery rate in shrimp samples reached 71-90% Determination of SGU residue in white leg shrimp samples: 0.16 ± 0.01ppm, banana prawn: 0.21±0.03ppm, greasyback shrimp: 0.12±0.02ppm, black tiger shrimp :0.26±0.02ppm, SMP residue in black tiger shrimp 0.09 ± 0.01 ppm No MTD, SDO, SMX, or SMP (except black tiger shrimp) were detected in the shrimp samples From the obtained results, we see that the HPLC - UV-Vis Detector method has high sensitivity, suitable for simultaneous analysis of metronidazole and antibacterial agents SGU, SMP, SDO, and SMX in shrimp Conclusion: We anticipate that these studies will advance the use of HPLC methods in general and UVVis, in particular, to determine metronidazole and sulfonamide in food to efficiently improve the science industry, particularly in the area of food safety and hygiene, helping to protect the health of humans 10| P a g e References Vietnamese Chu Đình Bính, Phạm Luận, Nguyễn Thị Ánh Nguyệt, 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derivatization”, Journal of Chromatography A, 726(1-2) 11| P a g e ... depend on the pH of buffer acetate 3.3.3 Ratio of mobile phase components The ratio of solvent components to the mobile phase affects the elution of the samples from the separation column As the mobile... height (H) or area (S) of the peek     H = k.Cb   or   S = k.Cb     In which H: height of the peek S: area of the peek k: experimental constant (depends on the conditions of the experiment) b: nature... ratio of mobile phase components 3.3.4 Speed of mobile phase The speed of the mobile phase is a decisive factor in the elution of substances in the chromatographic column, because it affects the