MSACL 2025 Abstract

Contamination in 3MTO Internal Standard Peaks in a Quantitative LC-MS/MS Plasma Metanephrine Assay

Sherrine Alleyne, Nicole Raut, Meagan Manual, Vijay Ramanaidoo
Department of Clinical Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust, North West London Pathology

Sherrine Alleyne, BSc Biomedical Science (Presenter) North West London Pathology

Presenter Bio: Hello my name is Sherrine, I have worked in Charing Cross Hospital in the Clinical Biochemistry Department for 25 years. I have been a senior in the LCMS laboratory since 2020 and have been involved in the implementations of 3 new WATERS MASS Specs; two TQ-S micro’s and a TQ-A Absolute.
I have extensive experience in Clinical diagnostics including Automation, Proteins, Endocrinology, HPLC and the Oncology Laboratories.
Since joining the Mass Spec laboratory, I have developed a greater interest in Mass Spec.
Taking part of the MSACL conference will allow me see other divisions and aspects of Mass Spectrometry, and the upcoming innovation involved with these platforms.

Relevant Financial Disclosures (within past 24 months, reported on Mar 24, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

1. Problem

A high level of background interference developed in the D4 3-methoxytyramine (3-MTO) Internal Standard (IS) transition (172.1>95.3) of our routine plasma metanephrines assay. This issue prevented the clinical reporting of 3-methoxytyramine.

2. Method Information
• 150 µl of patient plasma and 200 µl solution of D3 Metanephrine (MET), D3 Normetanephrine (NMET) and D4 3-MTO, IS solution mixed and extracted on a 96-well Oasis WCX µElution plate.
• Waters ACQUITY UPLC system
• Waters TQ-XS and TQ-S micro mass spectrometer
• Mobile Phase A: 100 mM ammonium formate, pH 3.0 with formic acid.
• Mobile Phase B : 100% acetonitrile (ACN)
• Waters Acquity UPLC BEH Amide 1.7um, with VanGuard Pre-Column
• UPLC gradient 2%A/98%B to 35%A/65%B over 4 minutes, then returns to the starting conditions , 2%A/98%B at 5.00 minutes at a flow rate of 0.20 ml/min & column temperature 35 oC.
• Injection volume: 15 µl on the TQ-XS and 5ul on the TQ-S micro.
• Quantitative SRM- MET – 180>148.3 NMET – 166.0>134.3 3MTO – 168.0>91.3

3. Troubleshooting Steps
Various troubleshooting steps were carried out to investigate the source of contamination.
Firstly, new internal standards made up and used in the assay, followed by a change of manufacturer for the ACN and the contamination was still present.

An Acid wash was carried out twice on the TQ-S micro, but each time the contamination was subsequently reintroduced into the system. After various experiments on the TQ-S micro it was established that the source of the contamination was mobile phase A. Therefore, new ammonium formate was ordered from a new supplier, but still the contamination remained. A new stock of the original supplier for the Ammonium formate was ordered and used in the mobile phase, but again still no change.

The in house LCMS grade H20 used for the mobile phase A was replaced with Honeywell bottled LCMS grade water, but there was still no change in the test assays.
Mobile phase A was made up with LCMS grade H20 and ammonium formate only which yielded a run without any contamination present. Thus the contamination was associated with the formic acid. Two other sources of formic acid were used to make up two separate mobile phase A, and surprisingly both runs showed contamination. When making up mobile phase A, a pH meter is used to titrate the pH of the mobile phase to 3, by slowly dripping the formic acid into the mobile phase using a plastic Pasteur pipette. Therefore, it was decided to not use a plastic Pasteur pipette and use a glass pipette to add the formic acid instead. When this was carried out and a test assay was run on both the TQ-XS and the TQ-S micro, no contamination was observed in the 3MTO IS peak. Although interference from the plastic Pasteur pipettes had not been observed previously, the laboratory had recently changed the supplier of these pipettes.

4. Outcome
Due to the plastic from the plastic Pasteur pipette leading to contamination of the formic acid, when carrying out the plasma metanephrines assay, plastic Pasteur pipettes are no longer used to avoid introducing the contamination to the assay and glass Pasteur pipettes are used instead.