MSACL 2023 Abstract

Background Interfering Peaks in a Quantitative LC-MS/MS Dihydrotesterone Assay Luisa Beltran, Sherrine Alleyne, Amrit Dhani, Louise Oliver, Emma Williams Department of Clinical Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust, North West London Pathology Luisa Beltran, BSc, MSc, PhD (Presenter) Imperial College Healthcare NHS Trust, North West London Pathology >> POSTER (PDF)

Presenter Bio: Senior Clinical Scientist working in Clinical Biochemistry, Imperial College Healthcare NHS Trust, North West London Pathology (UK).
My interest in mass spectrometry began whilst completing my PhD in Proteomics at Barts Cancer Institute, Queen Mary University of London. I then began my career in the clinical laboratory and throughout the years have worked with mass spectrometry in the areas of toxicology, inherited metabolic disease and endocrinology.
I am in training to complete The Royal College of Pathologists FRCPath examinations and working towards expanding the repertoire of the LC-MS/MS laboratory at North West London Pathology to better meet the needs of our patients.

1. Problem
High background and interfering peaks were observed in the analytical RT window for 13C3 dihydrotesterone (13C3-DHT) transition (294.5 > 258.5) in some patient samples, preventing the accurate integration of this internal standard (IS) and subsequent dihydrotesterone (DHT) quantification. This interference was not observed during the method validation and affected a small, but increasing, number of patient samples.

2. Method Information
• 150 µl of patient serum and 13C3-DHT IS solution mixed and extracted on a 96-well Oasis MAX µElution plate.
• Waters ACQUITY UPLC system
• Waters TQ-XS mass spectrometer
• Mobile Phase A: 0.05 mmol/L Ammonium Fluoride in H2O
• Mobile Phase B: 100% MeOH
• Cortecs® UPLCS® C18 1.6 µm 2.1 x 100 mm column with guard column
• UPLC gradient 60%A/40%B to 30%A/70%B over 3.5 min at a flow rate of 0.25 ml/min & column temperature 50oC.
• Injection volume: 15 µl
• Selective reaction monitoring (SRM) at 291.5>255.5 (DHT) and 294.5>258.5 (13C3-DHT).

3. Troubleshooting Steps
The intermittent nature of the interference did not suggest a system problem, but as a precaution all reagents were prepared from fresh stocks and the guard column was replaced, excluding them as the source of contamination.
Repeat analysis of affected and unaffected patient samples demonstrated that the interference was sample specific and did not exhibit carryover. An audit of 14 affected samples was conducted to identify common factors considering sample tube type, sample pathway, request location and patient characteristics. It was identified that all affected samples underwent automated aliquoting into false bottom polypropylene tubes (Sarstedt 62.617) on-board an Abbott a3600 Aliquoter Module. Experimental work demonstrated that incubation of serum in this tube type for 7 days at 4oC resulted in the presence of the interference whereas control serum incubated in a polypropylene test tube (GTIN: 5060443243032) under the same conditions was unaffected.

4. Outcome
Sample workflow was altered to avoid automated aliquoting into the affected tubes. The interference was not encountered again. This experience highlights the sensitivity of LC-MS/MS assays to sample workflow and consumable suppliers.