One-Stop Metabolite Shop

Purification and Metabolite Identification

Purification and Identification of Metabolites

Hypha have a wealth of expertise in the purification and identification of metabolites from complex matrices. We are able to purify drug metabolites from a variety of different matrices including from mammalian/human urine and plasma.

Our labs are well equipped with analytical UPLC-MS and HPLC-MS systems for high-throughput analysis and chromatographic method development. Our preparative HPLC systems include a mass-directed instrument and a number of UV-based Waters auto-purification systems, which enable purification of products ranging from sub milligram to multigram quantities. A range of evaporation technologies is available for sample concentration including centrifugal evaporation, for use at small scale, alongside rotary evaporation and freeze driers for lyophilisation. The chemistry laboratory is well set up to handle highly potent materials, with fume hoods and a balance enclosure for the safe handling of hazardous materials.

Hypha are also experts in the identification of metabolites using NMR spectroscopy. We use cryoprobe technology to gather data acquired on a 700 MHz machine, meaning only only small amounts of material are needed. We are familiar in interpreting spectra obtained from a wide variety of techniques for definitive identification of the structures of all types of metabolites.

Case Studies

Purification and identification of metabolites from a microbial biotransformation reaction

A glucuronide of a drug in clinical development was produced particularly well by a microbial biotransformation, with rapid conversion to the target product. A dose escalation study was conducted to optimise the reaction and the product glucuronide was extracted from the combined reaction mixtures and purified by a single step of preparative reversed phase HPLC yielding 136 mg with a purity of >99% by LC-ELSD. Some of this material was used for structure elucidation by NMR for definitive metabolite identification. The reaction was subsequently used again to produce a larger quantity of the target glucuronide and a stable-isotope-labelled analogue. A Certificate of Analysis was provided for both batches of material, including results of chromatographic analysis and GLP qNMR.

Purification of metabolites from urine

One purification project undertaken at Hypha resulted in tens of milligrams of the R- and S- O– glucuronides of carisbamate, a neuromodulator developed by SK Life Science, which were purified to >95% purity from 150 ml of urine using a three-step purification method. Confirmation of the structures was obtained by NMR spectroscopy, with qNMR enabling the use of the purified materials as analytical standards for bioanalysis. 

carisbamate metabolites case study image

Resources

Explore our library of resources comprising brochurescase studiesposters and publications about the work we do

Access to multiple metabolites needed to support clinical development is not always straightforward, and can sometimes mean that more than one technique needs to be applied to fulfil requirements. In one such project, a US pharma client required > 200 mg of three metabolites of a drug; an N-glucuronide (M1), an indirect O-glucuronide (M2b) and a hydroxylated metabolite (M8b). As part of this project, multiple components of Hypha’s one-stop metabolite shop were employed, including chemical synthesis, microbial biotransformation as well as purification and structure elucidation by NMR.

If clearance mechanisms of the test drug results in sufficient quantities of the major metabolites in biological material such as faeces or urine, purification and subsequent identification of metabolites from such matrices is possible. One such project undertaken at Hypha resulted in tens of milligrams of the R- and S-O-glucuronides of carisbamate, a neuromodulator developed by SK Life Science, which were purified to >95% purity from 150 ml of urine using a three-step purification method.

In Chapter 4 of the book on “Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters”, Hypha authors summarise the different methods employed for producing metabolites of drugs, illustrated with representative examples from the literature and work undertaken at Hypha. The chapter also includes a discussion and examples of the use of NMR spectroscopy for structure elucidation of metabolites.

Our other one-stop shop solutions for accessing and characterising metabolites

Hypha’s One-Stop Metabolite Shop enables synthesis, purification and characterization of all the main types of mammalian phase 1 and 2 metabolites.

Chemical Synthesis

Late-stage chemical glucuronidation

Mammalian biotransformation

Panels of liver S9s / microsomes

Test tubes with dripper
251A3171-DE01-48EB-AA27-BD82BC5AC10DCreated with sketchtool.

Hypha Discovery came highly recommended and we subsequently contracted with Hypha to extract and purify metabolites out of rabbit urine. As they were so successful with that extraction, we again requested help for a second program to extract metabolites from human urine and/or synthesize/biosynthesize 3 metabolites. As expected, Hypha has been successful preparing these metabolite reference standards along with structural elucidation and certificates of analysis. In addition, synthesis of one of the metabolites had been attempted at 2 other labs without success; however, Hypha was able to synthesize this difficult metabolite which allowed us to do further evaluations on the metabolite.

Head of Toxicology/DMPK

US Pharma Company

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