Isolation of High Purity Phase II Biotransformation Conjugates from Urine using HPLC fractionation: A Proof of Concept

Abstract

Biotransformation plays an essential role in converting xenobiotics into more excretable forms. Phase II biotransformation, also known as the conjugation phase, is often overlooked. Complications in phase II biotransformation activity can lead to severe adverse health consequences and even death. One of the methods for assessing biotransformation activity is by administering probe drugs and measuring the concentrations of the drug biotransformation conjugates excreted in urine after 12 hours using HPLC-UV/Vis or HPLC-MS. To produce sensitive and selective HPLC results, analytical standards are required during method development and for quality control. Analytical standards for some phase II biotransformation conjugates are very expensive and, in some cases, unavailable for purchase, as these compounds can be difficult to synthesise. An alternative approach to synthesising analytical standards would be to isolate them from a matrix containing high concentrations of these conjugates. The urine samples at the biotransformation and oxidative stress status (BOSS) laboratory serve as a good starting material since these urine samples already contain high concentrations of biotransformation conjugates. The aim of this study was to develop a method that can be used for the isolation of high-purity phase II biotransformation conjugates. Salicyluric acid and paracetamol glucuronide were selected as the metabolites of interest, as these metabolites are the phase II biotransformation end-products of aspirin and paracetamol metabolism. Hippuric acid as selected as an additional phase II biotransformation conjugate based on the polarity of the molecule and its role in benzoic acid metabolism. Based on the literature reviewed HPLC is the gold standard method for isolating small polar compounds, such as phase II biotransformation conjugates. HPLC has the added benefit of being able to be combined with an inline fraction collector, allowing for highly efficient fraction collection. During the first part of the study, a liquid chromatography fractionation method was developed for the isolation of high-purity phase II conjugates from a standard mixture. The method was divided into 1) sample pre-treatment, 2) chromatography optimisation and fraction collection, and 3) MS optimisation. In the second phase of the study, the effectiveness of the optimised extraction, LC-UV and fraction collection methods was tested. This was achieved by employing these methods on a urine sample to isolate hippuric acid, salicyluric acid and paracetamol glucuronide. Ethyl acetate liquid-liquid extraction proved to be most efficient and scalable for the isolation of hippuric acid, salicyluric acid and paracetamol glucuronide, with recoveries of 23.74 %, 32.17 % and 45.44 % respectively. Chromatographic separation on the Agilent XBD C18 (150 × 4.6 mm 5 μm) produced excellent chromatographic resolution that aided with easier fraction collection. Time-based fraction collection proved most effective for fraction collection, as abundance-based detection missed compounds below a specific threshold. MS source conditions and transition states were well optimised and could reliably detect hippuric acid, salicyluric acid and paracetamol glucuronide. The most abundant MRM transitions were as follows: 328.1 → 152 m/z for paracetamol glucuronide, 180.1 →105 m/z for hippuric acid, and 196.1 → 121 m/z for salicyluric acid. The optimised source conditions for these compounds were as follows: a drying gas temperature of 350°C, a drying gas flow rate of 3 L/min, a nebuliser pressure of 45 psi, and a capillary voltage of 6000 volts. Purity assessment of the primary fractions captured from urine using isocratic HPLC produced single peaks at the expected retention time for hippuric acid and salicyluric acid. The paracetamol glucuronide fraction captured was not pure after primary fractionation and required secondary fractionation. The secondary fractionation produced was a single pure peak indicating that HPLC fractionation can be employed for the isolation of high-purity phase II biotransformation conjugates.