Enhancing Biotransformation Identification Efficiency Using LC-MS Fine Isotope Structure Produced With Multi Reflecting Time-of-Flight MS

Potentially toxic drug metabolites can be difficult to detect and identify in the presence of complex matrices such as blood and urine. Using multiple analytical approaches to improve detection rates and identification confidence increases cost but more importantly slows, go, no-go decisions. Increasing LC-MS data specificity can help address these bottlenecks.

A UPLC-MS^E (ES+ and ES-) data independent acquisition (DIA) urinary screening assay was previously performed using a SELECT SERIES™ MRT at an acquisition rate of 10 Hz, with a system mass resolving power >200,000 FWHM. Precursor and fragment ion part per billion (ppb) accurate mass measurement facilitated the identification of metabolites of three therapeutic drugs present in a patient’s urine sample.

Further confirmatory investigations have been performed using ultra high mass resolution MS in combination with Ultra Performance LC™ (UPLC™). Resolution Enhancement Mode (REM) has a system resolving power >300,000 FWHM and delivers fine isotope structure (FIS) with ppb mass accuracy providing highly specific identification criterion and is a powerful tool to determine elemental composition and facilitate confident assignment of metabolite identifications. 

Benefits

Improved efficiency of drug metabolite workflows through increased mass resolving power, mass measurement accuracy and fine isotope structure at chromatographic timescales.

• Identify unknowns with increased confidence, reduce false detection rates and improve analysis efficiency
  
• Enhanced analyte detection in complex biological matrices using LC-MS and mass resolving power >300,000 FWHM
  
• Fine isotope structure distributions with ppb mass accuracy provides additional criteria to enhance identification confidence for small molecule therapeutic drugs and metabolites

High resolution mass spectrometry coupled with liquid chromatography is an ideal analytical tool for discovery and identification of drugs and their metabolites in complex biological matrices, aiding drug development workflows. Increasing mass resolution and mass accuracy subsequently increases confidence in compound assignment.

The SELECT SERIES MRT (Figure 1) is a state-of-the-art hybrid quadrupole Multi Reflecting Time-of-Flight Mass Spectrometer (MRT).¹ In MRT mode, where ions experience one pass of the MRT mass analyser, it provides a unique combination of high mass resolving power (>200,000 FWHM), and routine ppb mass accuracy, independent of acquisition speed.^2,3 In Resolution Enhancement Mode (REM), ions experience two passes of the MRT mass analyser, as a result a system mass resolving power of >300,000 FWHM is attained.

Using Ultra Performance LC coupled to a SELECT SERIES MRT (system resolution >200,000 FWHM), a non-targeted urinary screen of a healthy human volunteer was undertaken using ES+/ES- and has been fully described elsewhere.^2,3 Utilizing a metabolite identification workflow naproxen, carbamazepine, acetaminophen therapeutic drugs and metabolites, were identified. At >200,000 FWHM fine isotope structure was attained, providing added confidence when identifying biotransformation products, present in a complex biological matrix. In an extension to this study the samples were analyzed using REM, which provides the opportunity to generate mass spectra with enhanced fine isotope distribution information to facilitate identification of knowns and unknowns.

In this study we demonstrated the benefit of REM and present identified biotransformation products (Figure 2), including their resolved fine isotope distributions which incorporate ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S and ³⁴S.

Sample description

Human urine sample diluted 1:10 (H₂O)
Dosage: Carbamazepine (2 x 200 mg tablets), acetaminophen (2 x 500 mg tablets), naproxen (1 x 500 mg tablet).
Sample time points: 0, 2, 4, and 6 hours after medication was administered.

High mass resolution enhances ion selectivity of analytes in complex matrices. The number of possible elemental compositions generated with ppb high mass accuracy data is reduced and consequently there is a high degree of confidence in analyte identification. In conjunction with fine isotope structure, enhanced mass accuracy specificity can be utilised to improve identification certainty in research areas involving small molecules, such as metabolite identification.

The benefits of utilizing REM mode can be seen in Figure 3, where performing LC-MS (10 Hz) at >300,000 FWHM, for acetaminophen sulfate nine peaks are resolved for the combined A+1 and A+2 fine isotope distributions. Comparatively, six fine isotope peaks are resolved at 200,000 FWHM.

In Figure 4, the FIS is compared determined using 200,000 FWHM and 300,000 FWHM determined for the known acetyl-S-cysteine conjugate of acetaminophen. FIS provides additional identification specificity and in the case of the acetaminophen-acetyl-S-cysteine conjugate, a dual polarity FIS cluster can be generated. At 300,000 FWHM, for the combination of ES+/ES- A+1 and A+2 fine isotope distributions, a total of eighteen isotopic peaks are resolved and can be used to confirm biotransformation identity.

For the human urinary screen performed we have previously reported the identification of desmethyl naproxen sulfate ([M-H]^- m/z 295) and carbamazepine hydroxy sulfate metabolite ([M-H]^- m/z 331).^2,3 Both metabolites are rarely reported.^4-11 However the combination of ppb mass accuracy for precursor ion and fragments ions, in combination with FIS at 200,000 FWHM facilitated confident identification (see Figure 5). For these chromatographically coeluting (t_r 4.80) biotransformation products, REM data has been obtained (see Figure 6). The benefit of REM is illustrated once more, in Figure 6, for [desmethyl naproxen sulfate–H]^-, FIS resolution is enhanced and for [carbamazepine-o-sulfate - H]^- where for A+1 and A+2 combined, ten FIS peaks have been resolved. In the case of carbamazepine-o-sulfate, ppb mass accuracy has been obtained for seven fine isotope distribution peaks. These additional seven identification points add further confidence to the assignment of the detected biotransformation product. For the examples illustrated, sufficient intensity has been obtained to measure the A+1 and A+2 isotopic fine structure. In the case of [carbamazepine-o-sulfate - H]^-, the fine isotope peak “d”, was observed with a mass measurement error of 1.8 ppm, this is due to a contribution in the resultant centered data from fine isotope peak “c”. Utilising REM has also increased the certainty that the three components eluting at 4.66 min, 4.8 min and 5.28 min (Figure 6 (I)) are isomeric biotransformation products. At respective system resolutions the measured fine isotope structure peaks predominantly correspond to the positions of simulated isotopic distributions.¹²

Using the SELECT SERIES MRT, routine ppb mass accuracy is attained for precursor and fragment ions when performing complex urinary non-targeted screening analysis, with ES+ and ES- LC-MS at 10 Hz (system resolving power >200,000 FHWM). Utilizing the MRT REM mode the system resolving power is increased to >300,000 facilitating routine ppb mass accuracy of fine structure peak distributions.
For LC-MS acquisition at >200,000 and >300,000 FWHM the measured FIS peaks predominantly correspond to the positions of simulated isotopic distributions. Confidence in therapeutic drug xenobiotics and metabolite identification is enhanced where ppb mass accuracy is attained for mass resolved FIS structure in an LC-MS time frame. This FIS transformative mass measurement affords the opportunity to increase identification criterion options, in turn providing confidence to efficiently identify known and previously unknown biotransformation products.

Increased credence for metabolite identification is illustrated for the detection of phase II metabolite desmethyl naproxen sulfate coeluting with a sulfonated hydroxy carbamazepine, and isomeric biotransformation products.

MRT REM mass spectrometry affords the opportunity to harness multi-faceted benefits including utilising FIS enhanced specificity in data processing workflows, stable isotope labelling applications, and provide additional identification criterion where fragment ion information is absent for non-labile compounds.

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