Summary of Study ST003735

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002321. The data can be accessed directly via it's Project DOI: 10.21228/M8MJ9D This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003735
Study Title	Evaluation of in vitro pharmacodynamic drug interactions of ceftazidime-avibactam with tigecycline in ESBL- (extended spectrum beta-lactamase) and carbapenemase producing Escherichia coli
Study Summary	Background: Combination therapy offers a promising option to enhance efficacy and prevent resistance. A comprehensive and quantitative assessment of the last-resort combination of ceftazidime/avibactam and tigecycline is not available. Objective: This study systematically investigated the pharmacodynamic interaction between ceftazidime/avibactam and tigecycline in clinical and isogenic Escherichia coli strains harbouring genes that encode various carbapenemases or ESBLs (extended spectrum beta-lactamases). Methods: An adaptive in vitro 'dynamic' checkerboard design and pharmacometric modelling were employed for the evaluation of pharmacodynamic interactions in fifteen bacterial isolates. Additionally, time-kill assays and metabolomic analyses were used to provide mechanistic insights. Metabolomic analysis: Mechanistical investigation of the PD interaction between ceftazidime/avibactam-tigecycline was studied in a selected clinical isolate of E. coli (strain JUM_JEA) using metabolomic analyses in mono- and combination treatment scenarios. Time-kill assays were conducted for ceftazidime/avibactam and tigecycline concentrations of 4 x MIC (minimum inhibitory concentration) as well as combinations of both antibiotics at 4 x MIC CZA – 4 x MIC TGC and growth controls as four replicates over 4 h with samples at 0, 2 and 4 h. Results: Antagonistic drug interactions between ceftazidime/avibactam and tigecycline were identified in the majority of tested strains. Time-kill assays confirmed antagonistic interactions, with tigecycline limiting ceftazidime/avibactam total killing. Metabolomic analyses of mono and combined drug exposure to bacteria revealed matching metabolomes in tigecycline alone and the combination with ceftazidime/avibactam, corroborating the identified antagonism between these drugs.
Institute	European Molecular Biology Laboratory
Department	EMBL Heidelberg
Last Name	Drotleff
First Name	Bernhard
Address	Meyerhofstr. 1, Heidelberg, BW, 69117, Germany
Email	bernhard.drotleff@embl.de
Phone	none
Submit Date	2025-02-07
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-03-13
Release Version	1

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Project:

Project ID:	PR002321
Project DOI:	doi: 10.21228/M8MJ9D
Project Title:	Evaluation of in vitro pharmacodynamic drug interactions of ceftazidime-avibactam with tigecycline in ESBL- (extended spectrum beta-lactamase) and carbapenemase producing Escherichia coli
Project Summary:	Background: Combination therapy offers a promising option to enhance efficacy and prevent resistance. A comprehensive and quantitative assessment of the last-resort combination of ceftazidime/avibactam and tigecycline is not available. Objective: This study systematically investigated the pharmacodynamic interaction between ceftazidime/avibactam and tigecycline in clinical and isogenic Escherichia coli strains harbouring genes that encode various carbapenemases or ESBLs (extended spectrum beta-lactamases). Methods: An adaptive in vitro 'dynamic' checkerboard design and pharmacometric modelling were employed for the evaluation of pharmacodynamic interactions in fifteen bacterial isolates. Additionally, time-kill assays and metabolomic analyses were used to provide mechanistic insights. Metabolomic analysis: Mechanistical investigation of the PD interaction between ceftazidime/avibactam-tigecycline was studied in a selected clinical isolate of E. coli (strain JUM_JEA) using metabolomic analyses in mono- and combination treatment scenarios. Time-kill assays were conducted for ceftazidime/avibactam and tigecycline concentrations of 4 x MIC (minimum inhibitory concentration) as well as combinations of both antibiotics at 4 x MIC CZA – 4 x MIC TGC and growth controls as four replicates over 4 h with samples at 0, 2 and 4 h. Results: Antagonistic drug interactions between ceftazidime/avibactam and tigecycline were identified in the majority of tested strains. Time-kill assays confirmed antagonistic interactions, with tigecycline limiting ceftazidime/avibactam total killing. Metabolomic analyses of mono and combined drug exposure to bacteria revealed matching metabolomes in tigecycline alone and the combination with ceftazidime/avibactam, corroborating the identified antagonism between these drugs.
Institute:	European Molecular Biology Laboratory
Department:	EMBL Heidelberg
Last Name:	Drotleff
First Name:	Bernhard
Address:	Meyerhofstr. 1, Heidelberg, BW, 69117, Germany
Email:	bernhard.drotleff@embl.de
Phone:	none
Publications:	https://doi.org/10.1016/j.ijantimicag.2025.107457
Contributors:	Aneeq Farooq, Bernhard Drotleff, Niklas Kroemer, Mei-Ling Han, Jian Li, Jean Winoc Decousser, David Schrey, Julien Buyck, Nicolas Grégoire, Patrice Nordmann, Sebastian G. Wicha

Subject:

Subject ID:	SU003867
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562
Genotype Strain:	JUM_JEA

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Timepoint	Sample source
SA407528	Pellet_negX3_t0_2	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407529	Pellet_negX3_t0_4	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407530	Pellet_negX3_t0_3	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407531	Pellet_posX3_t0_4	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407532	Pellet_posX3_t0_3	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407533	Pellet_negX3_t0_1	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407534	Pellet_posX3_t0_1	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407535	Pellet_posX3_t0_2	0MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407536	Supernatant_negX3_t0_4	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407537	Supernatant_posX3_t0_4	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407538	Supernatant_negX3_t0_3	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407539	Supernatant_negX3_t0_2	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407540	Supernatant_negX3_t0_1	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407541	Supernatant_posX3_t0_1	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407542	Supernatant_posX3_t0_2	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407543	Supernatant_posX3_t0_3	0MIC CZA - 4MIC TGC	0h	E coli bacteria medium supernatant
SA407544	Pellet_posX3_t2_2	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407545	Pellet_posX3_t2_1	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407546	Pellet_negX3_t2_1	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407547	Pellet_negX3_t2_2	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407548	Pellet_negX3_t2_4	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407549	Pellet_negX3_t2_3	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407550	Pellet_posX3_t2_3	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407551	Pellet_posX3_t2_4	0MIC CZA - 4MIC TGC	2h	E coli bacteria cell pellet
SA407552	Supernatant_posX3_t2_2	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407553	Supernatant_posX3_t2_1	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407554	Supernatant_posX3_t2_3	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407555	Supernatant_posX3_t2_4	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407556	Supernatant_negX3_t2_4	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407557	Supernatant_negX3_t2_3	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407558	Supernatant_negX3_t2_2	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407559	Supernatant_negX3_t2_1	0MIC CZA - 4MIC TGC	2h	E coli bacteria medium supernatant
SA407560	Pellet_negX3_t4_1	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407561	Pellet_negX3_t4_2	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407562	Pellet_negX3_t4_3	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407563	Pellet_negX3_t4_4	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407564	Pellet_posX3_t4_4	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407565	Pellet_posX3_t4_1	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407566	Pellet_posX3_t4_3	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407567	Pellet_posX3_t4_2	0MIC CZA - 4MIC TGC	4h	E coli bacteria cell pellet
SA407568	Supernatant_posX3_t4_3	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407569	Supernatant_negX3_t4_1	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407570	Supernatant_negX3_t4_2	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407571	Supernatant_negX3_t4_4	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407572	Supernatant_posX3_t4_4	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407573	Supernatant_negX3_t4_3	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407574	Supernatant_posX3_t4_1	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407575	Supernatant_posX3_t4_2	0MIC CZA - 4MIC TGC	4h	E coli bacteria medium supernatant
SA407576	Pellet_posX2_t0_1	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407577	Pellet_negX2_t0_1	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407578	Pellet_negX2_t0_2	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407579	Pellet_negX2_t0_3	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407580	Pellet_negX2_t0_4	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407581	Pellet_posX2_t0_4	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407582	Pellet_posX2_t0_3	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407583	Pellet_posX2_t0_2	4MIC CZA - 0MIC TGC	0h	E coli bacteria cell pellet
SA407584	Supernatant_posX2_t0_2	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407585	Supernatant_posX2_t0_1	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407586	Supernatant_negX2_t0_3	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407587	Supernatant_negX2_t0_1	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407588	Supernatant_negX2_t0_4	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407589	Supernatant_posX2_t0_4	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407590	Supernatant_posX2_t0_3	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407591	Supernatant_negX2_t0_2	4MIC CZA - 0MIC TGC	0h	E coli bacteria medium supernatant
SA407592	Pellet_posX2_t2_2	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407593	Pellet_posX2_t2_1	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407594	Pellet_posX2_t2_3	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407595	Pellet_posX2_t2_4	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407596	Pellet_negX2_t2_4	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407597	Pellet_negX2_t2_3	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407598	Pellet_negX2_t2_2	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407599	Pellet_negX2_t2_1	4MIC CZA - 0MIC TGC	2h	E coli bacteria cell pellet
SA407600	Supernatant_negX2_t2_3	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407601	Supernatant_negX2_t2_1	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407602	Supernatant_posX2_t2_4	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407603	Supernatant_negX2_t2_2	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407604	Supernatant_posX2_t2_1	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407605	Supernatant_posX2_t2_2	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407606	Supernatant_posX2_t2_3	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407607	Supernatant_negX2_t2_4	4MIC CZA - 0MIC TGC	2h	E coli bacteria medium supernatant
SA407608	Pellet_posX2_t4_4	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407609	Pellet_posX2_t4_1	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407610	Pellet_posX2_t4_2	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407611	Pellet_posX2_t4_3	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407612	Pellet_negX2_t4_1	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407613	Pellet_negX2_t4_4	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407614	Pellet_negX2_t4_2	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407615	Pellet_negX2_t4_3	4MIC CZA - 0MIC TGC	4h	E coli bacteria cell pellet
SA407616	Supernatant_posX2_t4_2	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407617	Supernatant_negX2_t4_4	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407618	Supernatant_negX2_t4_3	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407619	Supernatant_negX2_t4_2	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407620	Supernatant_negX2_t4_1	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407621	Supernatant_posX2_t4_3	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407622	Supernatant_posX2_t4_4	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407623	Supernatant_posX2_t4_1	4MIC CZA - 0MIC TGC	4h	E coli bacteria medium supernatant
SA407624	Pellet_posX4_t0_2	4MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407625	Pellet_negX4_t0_3	4MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407626	Pellet_posX4_t0_1	4MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet
SA407627	Pellet_posX4_t0_4	4MIC CZA - 4MIC TGC	0h	E coli bacteria cell pellet

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Collection:

Collection ID:	CO003860
Collection Summary:	Time-kill assays were performed by inoculating and pre-incubating the bacterial isolate (approx. 1 x 10^6 CFU/mL) in ca-MHB for 2 h at 37 °C ambient air before adding ceftazidime/avibactam (CZA) and/or tigecycline (TGC). For bacterial cell pellet analysis, samples were quenched in an ethanol/dry ice bath for 30 s, centrifuged at 3,220 x g for 10 min to obtain cell pellets. After separating the supernatant (250 μL per sample), cell pellets were resuspended twice in 0.5 mL of 4 °C saline, centrifuged at 2,576 x g for 3 min, and stored at -80 °C until analysis.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR003876
Treatment Summary:	Mechanistical investigation of the PD interaction between ceftazidime/avibactam-tigecycline was studied in a selected clinical isolate of E. coli (strain JUM_JEA) using metabolomic analyses in mono- and combination treatment scenarios. Time-kill assays were conducted for CZA and TGC concentrations of 4 x MIC as well as combinations of both antibiotics at 4 x MIC CZA – 4 x MIC TGC and growth controls as four replicates over 4 h with samples at 0, 2 and 4 h.

Treatment ID:

TR003876

Treatment Summary:

Mechanistical investigation of the PD interaction between ceftazidime/avibactam-tigecycline was studied in a selected clinical isolate of E. coli (strain JUM_JEA) using metabolomic analyses in mono- and combination treatment scenarios. Time-kill assays were conducted for CZA and TGC concentrations of 4 x MIC as well as combinations of both antibiotics at 4 x MIC CZA – 4 x MIC TGC and growth controls as four replicates over 4 h with samples at 0, 2 and 4 h.

Sample Preparation:

Sampleprep ID:	SP003873
Sampleprep Summary:	The methodology for sampling and preparation was adapted from the protocol described by Han et al. [DOI: 10.1128/AAC.02656-17]. For liquid chromatography-mass spectrometry (LC-MS/MS) analysis cell pellets were resuspended in 300 µL of a cold chloroform/methanol/water extraction solvent (1:3:0.9 v/v/v) augmented with isotope labelled amino acids (MSK-A2-1.2; Cambridge Isotope Laboratories, MA, USA) as internal standards at a concentration of 0.5% in the final sample for untargeted metabolomics.To permeabilize the cells and facilitate the release of intracellular metabolites, the resuspended mixtures were subjected to a freeze-thaw cycle. This process involved freezing the mixture in liquid nitrogen followed by thawing on ice, which was repeated three times. Subsequently, the mixture was maintained on ice water for 15 min and vigorously resuspended using a vortex mixer. The obtained samples as well as the extracellular supernatant samples were then centrifuged with 300 µL of each sample at 14,000 x g and 4 °C for 10 min. A total of 50 μL of the particle-free supernatant were then extracted via addition of 200 µL methanol (including internal standard). Samples were thoroughly vortexed and incubated for 20 min at -20 °C. After centrifugation for 10 min at 15,000 × g and 4 °C with a 5415R microcentrifuge (Eppendorf, Hamburg, Germany), supernatants were transferred and dried under a stream of nitrogen (Organomation Microvap, MA, USA). Dried samples were reconstituted in 50 µL of 80% methanol (v:v), vortexed, and centrifuged (see conditions described above). Ultimately, samples were transferred to silanized glass vials and directly injected into the analytical system.

Sampleprep ID:

SP003873

Sampleprep Summary:

The methodology for sampling and preparation was adapted from the protocol described by Han et al. [DOI: 10.1128/AAC.02656-17]. For liquid chromatography-mass spectrometry (LC-MS/MS) analysis cell pellets were resuspended in 300 µL of a cold chloroform/methanol/water extraction solvent (1:3:0.9 v/v/v) augmented with isotope labelled amino acids (MSK-A2-1.2; Cambridge Isotope Laboratories, MA, USA) as internal standards at a concentration of 0.5% in the final sample for untargeted metabolomics.To permeabilize the cells and facilitate the release of intracellular metabolites, the resuspended mixtures were subjected to a freeze-thaw cycle. This process involved freezing the mixture in liquid nitrogen followed by thawing on ice, which was repeated three times. Subsequently, the mixture was maintained on ice water for 15 min and vigorously resuspended using a vortex mixer. The obtained samples as well as the extracellular supernatant samples were then centrifuged with 300 µL of each sample at 14,000 x g and 4 °C for 10 min. A total of 50 μL of the particle-free supernatant were then extracted via addition of 200 µL methanol (including internal standard). Samples were thoroughly vortexed and incubated for 20 min at -20 °C. After centrifugation for 10 min at 15,000 × g and 4 °C with a 5415R microcentrifuge (Eppendorf, Hamburg, Germany), supernatants were transferred and dried under a stream of nitrogen (Organomation Microvap, MA, USA). Dried samples were reconstituted in 50 µL of 80% methanol (v:v), vortexed, and centrifuged (see conditions described above). Ultimately, samples were transferred to silanized glass vials and directly injected into the analytical system.

Combined analysis:

Analysis ID	AN006128	AN006129
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Waters Atlantis Premier BEH Z-HILIC (2.1 mm x 100 mm, 1.7 µm)	Waters Atlantis Premier BEH Z-HILIC (2.1 mm x 100mm, 1.7 μ"m)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 240	Thermo Orbitrap Exploris 240
Ion Mode	NEGATIVE	POSITIVE
Units	Peak area	Peak area

Chromatography:

Chromatography ID:	CH004653
Chromatography Summary:	LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative and positive ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1 mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate, when analysing in negative mode, and 10 mM ammonium formate, when analysing in positive mode. The aqueous portion of each mobile phase was pH-adjusted (negative mode: pH 9.0 via addition of ammonium hydroxide; positive mode: pH 3.0 via addition of formic acid). The following gradient (20 min total run time including re-equilibration) was applied (time [min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40 °C, the autosampler was set to 4 °C and sample injection volume was 5 µL.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis Premier BEH Z-HILIC (2.1 mm x 100 mm, 1.7 µm)
Column Temperature:	40
Flow Gradient:	time [min]/%B: 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95
Flow Rate:	0.25mL/min
Solvent A:	90% water/10% acetonitrile; 10 mM ammonium acetate
Solvent B:	90% acetonitrile/10% water; 10 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH004654
Chromatography Summary:	LC-MS/MS analysis was performed on a Vanquish UHPLC system coupled to an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA) in negative and positive ESI (electrospray ionization) mode. Chromatographic separation was carried out on an Atlantis Premier BEH Z-HILIC column (Waters, MA, USA; 2.1 mm x 100 mm, 1.7 µm) at a flow rate of 0.25 mL/min. The mobile phase consisted of water:acetonitrile (9:1, v/v; mobile phase A) and acetonitrile:water (9:1, v/v; mobile phase B), which were modified with a total buffer concentration of 10 mM ammonium acetate, when analysing in negative mode, and 10 mM ammonium formate, when analysing in positive mode. The aqueous portion of each mobile phase was pH-adjusted (negative mode: pH 9.0 via addition of ammonium hydroxide; positive mode: pH 3.0 via addition of formic acid). The following gradient (20 min total run time including re-equilibration) was applied (time [min]/%B): 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95. Column temperature was maintained at 40 °C, the autosampler was set to 4 °C and sample injection volume was 5 µL.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis Premier BEH Z-HILIC (2.1 mm x 100mm, 1.7 μ"m)
Column Temperature:	40
Flow Gradient:	time [min]/%B: 0/95, 2/95, 14.5/60, 16/60, 16.5/95, 20/95
Flow Rate:	0.25mL/min
Solvent A:	90% water/10% acetonitrile; 10 mM ammonium formate pH3
Solvent B:	90% acetonitrile/10% water; 10 mM ammonium formate pH3
Chromatography Type:	HILIC

MS:

MS ID:	MS005834
Analysis ID:	AN006128
Instrument Name:	Thermo Orbitrap Exploris 240
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data dependent acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: -3500 V (negative mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350 °C, vaporizer temperature: 300 °C.
Ion Mode:	NEGATIVE

MS ID:	MS005835
Analysis ID:	AN006129
Instrument Name:	Thermo Orbitrap Exploris 240
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Analytes were recorded via a full scan with a mass resolving power of 120,000 over a mass range from 60 – 900 m/z (scan time: 100 ms, RF lens: 70%). To obtain MS/MS fragment spectra, data dependent acquisition was carried out (resolving power: 15,000; scan time: 22 ms; stepped collision energies [%]: 30/50/70; cycle time: 900 ms). Ion source parameters were set to the following values: spray voltage: 4100 V (positive mode), sheath gas: 30 psi, auxiliary gas: 5 psi, sweep gas: 0 psi, ion transfer tube temperature: 350 °C, vaporizer temperature: 300 °C.
Ion Mode:	POSITIVE