Ultrasound-Assisted Extraction Combined with HPLC-UV for Fast Determination of Sulfamethazine and Its N₄-Acetyl Metabolite in Plasma and Phosphate Buffer

Abstract

A simple and cost effective HPLC-UV method for fast determination of sulfamethazine (SMZ) and its N₄-acetyl metabolite, N₄-acetyl sulfamethazine (AcSMZ), in plasma and phosphate buffer was developed and validated. The sample treatment was performed by ultrasound-assisted extraction with 1 mL ethyl acetate twice, each time 5 min. Moreover, there was no need for centrifugation and further clean-up, which shortened the measurement time. The total analysis time was less than 30 min. The method proved to be a selective and accurate tool for detection of SMZ and AcSMZ in plasma and phosphate buffer with satisfactory recoveries (85.43–99.29%) and little matrix effects (0.86–0.99). The proposed approach was also applied to the evaluation of pharmacokinetic and drug-metabolite protein binding interaction studies. The results show that SMZ and AcSMZ are non-concentration-dependent, and the metabolite does not bind competitively with the drug to the plasma protein.

Keywords:

• HPLC-UV
• N₄-acetyl sulfamethazine
• Phosphate buffer
• Plasma
• Sulfamethazine

Acknowledgments

The authors gratefully acknowledge the financial supports by National Natural Science Foundation of China (Grant No. 30972442), Natural Science Foundation of Tianjin (Grant No. 09JCYBJC08300), National Key Project of Scientific and Technical Supporting Programs (Grant NO. 2011BAK10B06), and State Key Laboratory of Envieonmental Chemistry and Ecotoxicoloay, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (Grant No. KF2009-15).

Notes

Note. The extraction efficiency is calculated from the recovery of the target compounds in the fortified plasma at SMZ for 20 µg/mL and AcSMZ for 10 µg/mL in relation to the same concentration of the standard in acetonitrile.

^a The recovery rates are expressed as Mean ±SD.

^a RSD, relative standard deviation.

^b LOD, limit of detection.

^c LOQ, limit of quantification.

Note. The concentration is expressed as Mean ± SD.

Notes. The values are expressed as Mean ± SD.

^a The protein binding rate is expressed as Mean ±SD.