Determination of p-aminohippuric acid with b- cyclodextrin sensitized fluorescence spectrometry† Rashed H. Alremeithi, ab Mohammed A. Meetani, * a Mu'ath K. Mousa, a Na'il I. Saleh a and John Graham a Sensitive spectrofluorometric and liquid chromatography with fluorescence detection methods have been developed for detection and determination of para-aminohippuric acid (PAH), one of the commonly used markers for estimating effective renal plasma flow, in the presence of b-cyclodextrin (b-CD). Fluorescence signals have been enhanced with the addition of b-CD to the drug aqueous solution. The 1 : 2 (host–guest) inclusion between PAH and cyclodextrin was evident by mass spectrometry and density functional theory (DFT) calculations supporting the formation of an excimer state at 355 nm. Factors that affect the PAH interaction with cyclodextrin have been investigated such as the size of the cyclodextrin cavity, the concentration of PAH, the concentration of cyclodextrin and pH effects. A calibration curve was established for the spectrofluorometric data of PAH with b-CD in the concentration range of 0.05–100 mM of PAH and the detection limit was 0.015 mM. HPLC with fluorescence detection was investigated in the presence of b-CD in the mobile phase. It was found that the calibration curve slope increased as the concentration of b-CD increased. Finally urine samples were spiked with 100 mM and 500 mM of PAH and showed recoveries in the range of 104–118% and 99.2–103%, respectively. Introduction The correct determination of effective renal plasma ow (ERPF) is crucial to evaluate kidney function in clinical or research settings. One of the commonly used markers for estimating ERPF is para-amino hippuric acid (PAH), since it is freely ltered at the glomerulus and undergoes extensive secretion and negligible reabsorption within renal tubules when it has low plasma concentration. 1 Many analytical methods have been established to measure PAH in plasma and urine, such as UV/vis spectroscopy, 2 HPLC with UV detec- tion, 3–8 electrochemical detections 9 and tandem mass spec- trometry. 10,11 Although PAH shows uorescence activity, few reports have studied the determination of PAH using HPLC- uorescence detection. 12 In the last two decades the macro cyclic oligosaccharides cyclodextrins, which consist of glucopyranose units attaching together, were reported to form enormous host–guest inclusion complexes, enhancing the analytical signal of the sequestrated guest molecules and therefore enhancing the sensitivity of their analytical detections in aqueous media. When the guest mole- cules are non-covalently encapsulated inside the cyclodextrin cavity, a modication of their chemical and physical properties always occurs due to the altered microenvironment, as well as connement and isolation from the surrounding medium such as the enhancement in solubility and uorescence emis- sion. 13–15 As a result, the use of cyclodextrins in improving drug solubility and stability and in particular in analytical sensing has increased in popularity. Aimed at detection and determination of PAH utilizing the supramolecular approach of b-CD, a sensitive method is developed here using two techniques; spectrouorometry and liquid chromatography with uorescence detection. Fluores- cence signals were enhanced with the addition of b-CD in aqueous solutions. The experimental conditions that gave the best results were investigated in terms of cyclodextrin cavity size, concentration of PAH, concentration of cyclodextrin, and pH effects. The interaction between PAH and cyclodextrin was investigated and considered as a host–guest inclusion which was evident by 1 H-NMR, mass spectrometry and DFT calcula- tions. A calibration curve was established from the spectrou- orometric HPLC with uorescence detection data. Finally urine samples were spiked a known amount of PAH and recoveries were calculated. a Chemistry Department, College of Science, United Arab Emirates University, P. O. Box 15551, Al-Ain, United Arab Emirates. E-mail: MMeetani@uaeu.ac.ae; Fax: +971 3 713 4928; Tel: +971 3 713 6136 b General Department of Forensic Science and Criminology, Dubai Police, Dubai, United Arab Emirates † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11742b Cite this: RSC Adv. , 2016, 6, 114296 Received 5th May 2016 Accepted 28th November 2016 DOI: 10.1039/c6ra11742b www.rsc.org/advances 114296 | RSC Adv. , 2016, 6, 114296–114303 This journal is © The Royal Society of Chemistry 2016 RSC Advances PAPER