LC-MS/MS MetID Service

Hypha offers LC-MS/MS based MetID to complement our existing definitive MetID capabilities using NMR spectroscopy. This allows MetID strategy, structural confirmation, and material generation to be addressed within a single, coherent workflow with us at Hypha. 

We support:

• In-depth biotransformation studies using multi-species hepatocytes, microsomes and related systems, including systems for slowly metabolised compounds
• LC-MS/MS-based metabolite identification to guide prioritisation and strategy
• Definitive structure elucidation at microgram scale using cryoprobe NMR spectroscopy
• Identification of metabolic soft spots to inform medicinal chemistry and risk assessment
• Milligram-gram scale of metabolites of all types to support downstream studies using biotransformation or chemical synthesis approaches

Free metabolite synthesis screening

For full Met-ID projects, we uniquely offer complementary PolyCYPs and PolyUGTs screening, which we have seen to provide additional context when advising clients on metabolite generation strategies, whether the goal is definitive identification or scaling material for downstream studies such as synthesis, safety assessment, or quantitative bioanalysis of metabolites.

We can tailor the MetID package to your requirements, contact us to find out more.

Webinar - save the date!

We'll be hosting a webinar on Thursday 19th March 2026 on how Hypha uniquely integrates LC-MS/MS-based Met-ID with definitive NMR confirmation and metabolite synthesis. Keep an eye out for invitations going out in February, or let us know if you're interested in attending and we'll add you to the list!

Instrumentation

We utilise the Thermo Scientific Vanquish Horizon UHPLC and Orbitrap Exploris^TM 120 LC-MS/MS instrumentation, which enables the detection and identification of a wide spectrum of both expected and unexpected metabolites, including those present at low abundance. Its sub-ppm mass accuracy and high resolution allow for differentiation of isobaric compounds, providing high confidence in metabolite identification.

We also routinely provide NMR spectroscopy based definitive MetID for those metabolites where knowledge of the full structure is important. A 700 MHz instrument fitted with a 1.7mm cryoprobe enables acquisition of a full data set using only small amounts of purified metabolite for structure elucidation. We also use a 600 MHz instrument with a special pentacryoprobe for enhanced C, N and F detection capabilities. Interpretation and reporting of the data is done by experts at Hypha.

Case study - MetID of repaglinide

Metabolism of repaglinide was studied in human and rat cryopreserved hepatocytes over 120 mins, with subsequent analysis by LC-MS/MS in positive and negative ionisation modes. Metabolites observed were compared to those reported in the literature, confirming production in Hypha’s incubation system. In addition, potential new metabolites were detected that have not been previously published.

In human hepatocytes, 14 metabolites were detected. The 3 main in vitro metabolites reported by Gan et al. (M2, M4 and the glucuronide M7) corresponded to the top 3 metabolites (HD14, HD8, HD7) observed in this study. Ten other metabolites were annotated, arising from oxidation of the aliphatic side chain, oxidative dehydrogenation, further oxidations and secondary glucuronidation. 

The metabolite map shows metabolite numbers assigned by Hypha with % MS peak response (+ve, -ve) together with the corresponding metabolite nomenclature reported in the literature.

Reference: Gan et al. Br J Clin Pharmacol. 2010; 70(6): 870-880. doi:10.1111/j.1365-2125.2010.03772.x M2, M4 and repaglinide glucuronide are reported to be the major metabolites produced by human hepatocytes in this paper.

Incubation of repaglinide with PolyCYPs and PolyUGTs reproduced the major metabolites produced in human hepatocytes (CYP derived metabolites M0-OH, M1, M2, M4, and the acyl glucuronide M7). Max conversions based on MS peak area (+ve mode) indicated good scalability, ranging from 7.6% to 83%.

This exemplifies the unique nature of Hypha's MetID service - we not only provide the metabolite data, but also an immediate route to synthesise key metabolites as soon as the need is identified, with the added benefit of definitive structure elucidation by NMR.

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