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Simultaneous Analysis of Sulfa Drugs

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Simultaneous Analysis of Sulfa Drugs TSKgel® TECHNICAL INFORMATION SHEET No. 118 No.11 Simultaneous Analysis of Sulfa Drugs Sulfa drugs are synthetic antibacterial agents that are widely used in the animal industry. On November 29, 2005, the Ministry of Health, Labor and Welfare of Japan issued the "Test methods for residual pesticides in foods, feed additives and veterinary drug components (partial revision)" (Shokuan No. 1129002). According to this document, many sulfa drugs are designated as compounds that require testing and standard values have been established. Here, as a mobile phase, formic acid and methanol were used and different sulfa drugs were analyzed simultaneously. Using a TSKgel ODS-100V, 3µm column it is possible to obtain a chromatogram with favorable peaks for highly polar substances such as sulfaguanidine. Figure 1. Chromatogram for sulfa drugs (each: 2.5mg/L) 3 2 4 13 5 7 9 11 16 1 6 10 15 17 12 14 18 19 8 0 5 10 15 20 25 30 RetentionRetention timetime ((min)minutes) Table 1. Conditions Column: TSKgel ODS-100V, 3µm, 4.6mm ID x 15cm A: 0.1% formic acid in water Mobile phase: B: 0.1% formic acid in methanol Gradient: 0min (0%B)→ 30min (70%B) →32min (70%B)→ 33min (0%B) Flow rate: 1.0mL/min Temperature: 40℃ Injection vol.: 10μL Samples: 1. sulfaguanidine 11. sulfamethoxypyridazine 2. sulfanilamide 12. sulfachloropyridazine 3. sulfacetamide 13. sulfamethoxazole 4. sulfadiazine 14. sulfamonomethoxine 5. sulfathiazole 15. sulfadoxine 6. sulfapyridine 16. sulfabenzamide 7. sulfamerazine 17. sulfadimethoxine 8. trimethoprim 18. sulfaquinoxaline 9. sulfamethizole 19. sulfanitran 10. sulfadimidin TSKgel® TECHNICAL INFORMATION SHEET No. 120 TOSOH Concurrent Analysis of Sulfa Drugs by LC/MS Sulfa drugs are synthetic antibacterial agents that are widely used in the animal industry. A notice issued by the Ministry of Health, Labor and Welfare [Shokuan No. 1129002; "Concurrent test method I for veterinary drugs by HPLC (animal husbandry and fishery)"] lists 16 sulfa drugs and specifies that a quantitative test must be performed by HPLC and a confirmation test must be performed by LC/MS(/MS). With regard to sulfa drug analysis, a past report (TSKgel Technical Information Sheet No. 118) introduced isolation using a UV detector. The present report measured the above-mentioned 16 sulfa drugs and trimethoprim (synthetic antibacterial agent often co-administered with sulfa drugs) using a MS detector. With all sulfa drugs, sharp peaks without tailing were obtained. For all sulfa drugs, quantification was confirmed within a concentration range of 0.02-1.0mg/L. Table 1: Sulfa Drugs Drug m/z sulfaguanidine 215 sulfacetamide 215 sulfadiazine 251 sulfathiazole 256 sulfapyridine 250 sulfamerazine 265 trimethoprim 291 sulfadimidin 279 sulfamethoxypyridazine 281 sulfamonomethoxine 281 sulfachloropyridazine 285 sulfamethoxazole 254 sulfadoxine 311 sulfadimethoxine 311 sulfabenzamide 277 sulfaquinoxaline 301 sulfanitran 336 Table 2. Measurement conditions HPLC Conditions Column: TSKgel ODS-100V, 3μm, 2.0mm ID x 15cm A: 0.1% formic acid Mobile phase: B: 0.1% formic acid in methanol Gradient: 0min (0%B) 20min (70%B) 22min (70%B) 23min (0% B) Flow rate: 0.2mL/min Temperature: 40℃ Injection vol.: 2μL MS Conditions ® MS: QTRAP (Applied Biosystems) Ion Source: ESI positive Mode: SIM Temperature: 500℃ Ionspray voltage: 5000 V Figure 1. SIM chromatogram of each sulfa drug (0.5mg/L） ) 2.0E+06 3.0E+06 m/z 215 m/z 281 cps 1 ( 2.0E+06 9 1.0E+06 2 1.0E+06 10 cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min 2.0E+06 1.0E+06 ) m/z 251 3 m/z 285 cps 11 ( 5.0E+05 1.0E+06 Intensity, cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min ) 2.0E+06 2.0E+06 m/z 256 cps m/z 254 ( 1.0E+06 4 1.0E+06 12 Intensity, cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min 3.0E+06 3.0E+06 ) m/z 250 2.0E+06 5 m/z 311 cps 2.0E+06 ( 1.0E+06 13 1.0E+06 14 cps Intensity, 0.0E+00 Intensity, cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min 2.0E+06 2.0E+06 ) m/z 265 6 m/z 277 cps ( 1.0E+06 1.0E+06 15 Intensity, cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min 4.0E+06 1.5E+06 ) m/z 291 7 m/z 301 cps 1.0E+06 ( 2.0E+06 16 5.0E+05 cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time, min 3.0E+06 4.0E+04 ) m/z 279 m/z 336 cps 2.0E+06 ( 8 2.0E+04 1.0E+06 17 Intensity, cps Intensity, 0.0E+00 cps Intensity, 0.0E+00 Intensity 0 5 10 15 20 25 0 5 10 15 20 25 Time, min Time (minutes) TimeTime, (min minutes) Samples: 1. sulfaguanidine 10. sulfamonomethoxine 2. sulfacetamide 11. sulfachloropyridazine 3. sulfadiazine 12. sulfamethoxazole 4. sulfathiazole 13. sulfadoxine 5. sulfapyridine 14. sulfadimethoxine 6. sulfamerazine 15. sulfabenzamide 7. trimethoprim 16. sulfaquinoxaline 8. sulfadimidin 17. sulfanitran 9. sulfamethoxypyridazine 2 .
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