Qtrap 5500 mass spectrometer

Liquid chromatography was performed on an Agilent Technologies (Santa Clara, CA) Infinity 1290 series HPLC system. The mass spectrometry analysis was performed on a SCIEX (Framingham, MA) 6500⁺ QTRAP mass spectrometer. The mass spectrometer analysis method utilized a positive MRM scan (Supplementary Tables S2 and S3). A SCIEX 5500 QTRAP mass spectrometer and SCIEX 4500 mass spectrometer were also used for certain aspects of the study.

Plasma tenofovir concentrations were determined with a validated liquid chromatography–tandem mass spectrometry assay developed at the Division of Clinical Pharmacology, University of Cape Town. The method utilized plasma protein precipitation, followed by high‐performance liquid chromatography with tandem mass spectrometry detection. Chromatographic separation was achieved on a Waters Atlantis T3 column (2.1 mm × 100 mm, 3 μm) with a total runtime of 6 min. A Sciex 5500 Qtrap mass spectrometer at unit resolution in the multiple reaction monitoring mode was used to monitor the transition of the protonated precursor ions, 288.1 and 294.1 to the product ions 176.1 and 182.1 for tenofovir and tenofovir‐d6 (internal standard), respectively. Electrospray ionization was used for ion production. The calibration curve fitted a quadratic (weighted by 1/concentration) regression based on peak area ratios over the range of 0.500 to 300 ng/mL. The combined accuracy (%Nom) of the limit of quantification, low, medium, and high‐quality controls (three validation batches, N = 18) were between 93.8% and 103.8%, with precision (percent coefficient of variation) less than 13%.

HPLC-MS/MS system consisted of Shimadzu Nexera High Performance Liquid Chromatograph and 5500 QTRAP mass spectrometer (Sciex, Framingham, MA). Chromatographic separation was achieved using a Kinetex C18 column (2.1 × 100 mm, 2.6 μm, Phenomenex, Torrance, CA) at 40°C. The mobile phase was composed of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B). The gradient elution program was performed as follows: 0.01–1 min, 15% B; 1–10 min, 15 to 60% B; 10–12 min, 95% B; 12–17 min, 15% B. The flow rate was 0.3 mL/min and the injection volume was 10 μL. The mass spectrometer was operated in negative mode for OTα, and positive mode for OTA, 2’R-OTA, OTB, and ¹³C₂₀—OTA. Quantification was achieved using multiple reaction monitoring (MRM) mode in one chromatographic run. The mass spectrometry was performed with electrospray ionization (ESI) interface at 500°C with the following settings: curtain gas: 25 psi, collision gas: medium, ionization voltage: −4,000 V (negative polarity) or +4,500 V (positive polarity), nebulizer gas: 60 psi, auxiliary gas: 60 psi. The optimization of compound dependent parameters, such as the declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP), was performed by flow injection analysis (Table 1S Supplementary material). Analyst 1.6.2 software was used for data acquisition and processing.