MSACL 2023 Abstract

The Importance of Using Isotopically Matched Internal Standards in Liquid Chromatography-mass Spectrometry-based Assays to Minimize Matrix Effects Karen Bolhuis, Katherine A. Turner, Micah Stoutjesdyk Spectrum Health Hospitals Karen Bolhuis, Bachelor of Science in Chemistry (Presenter) Corewell Health Hospitals

Presenter Bio: I am the Technical Specialist for the Corewell Health Toxicology Laboratory. I am responsible for regulatory upkeep as well as method development.

1. Problem
a. In the Forensic Toxicology, Criminalistics FTC-B 2021 College of American Pathologists (CAP) proficiency testing (PT) survey FTC-10 hydroxyzine was present. Our level reported for hydroxyzine was inconsistent with our peers, resulting in a +2.8 standard deviation index (SDI) from the mean, and we received an unacceptable score for this analyte.
b. In the validation of o-desmethylvenlafaxine, matrix effects were analyzed using 10 different plasma and serum samples negative for o-desmethylvenlafaxine. The matrix effect program in the MassLynx software was used that included post column infusion of the analyte and internal standard (IS). The program then calculates an IS normalized factor and coefficient of variable (CV). The matrix effect study resulted in an IS normalized factor of 3.58 with a 50.89% CV. This result failed to meet our acceptance criteria of 0.9-1.1 for the IS normalized factor and ≤15% CV.

2. Method Information
a. Hydroxyzine
i. Extract 100 µL of plasma or serum via protein precipitation utilizing zinc sulfate
ii. Waters Xevo TQMS with Acquity UPLC System
iii. Acquity UPLC 2.1x50 mm, BEH C18, 1.7 µm column
iv. Mobile phase A: 2 mM Ammonium Acetate in Water, 0.1% Formic Acid
v. Mobile phase B: 2 mM Ammonium Acetate in Methanol, 0.1% Formic Acid
vi. 3 min gradient LC program, 0.8 mL/min flow rate
vii. Column oven 55 °C
viii. Injection volume 5 µL
ix. Quantitative multiple-reaction monitoring mode (MRM) acquisition
b. O-desmethylvenlafaxine
i. Extract 100 µL of plasma or serum via protein precipitation utilizing zinc sulfate
ii. Waters Xevo TQ-S with Acquity UPLC System
iii. Acquity UPLC 2.1x50 mm, BEH C18, 1.7 µm column
iv. Mobile phase A: 2 mM Ammonium Acetate in Water, 0.1% Formic Acid
v. Mobile phase B: 2 mM Ammonium Acetate in Methanol, 0.1% Formic Acid
vi. 5 min gradient LC program, 0.6 mL/min flow rate
vii. Column oven 55 °C
viii. Injection volume 0.5 µL
ix. Quantitative multiple-reaction monitoring mode (MRM) acquisition

3. Troubleshooting Steps
a. To evaluate the unacceptable result for hydroxyzine in the FTC survey, the data from the analysis was reviewed. Issues with extraction, linearity, and chromatography were ruled out. Upon reviewing the raw data, it appeared the IS was suppressed. Acetopromazine is utilized as the IS for the quantitation of hydroxyzine. It has a retention time (RT) of 1.30 minutes, while hydroxyzine has a RT of 1.34 minutes. An isotopically matched IS, Hydroxyzine-13C6, was purchased from Cerilliant Corporation. Utilizing hydroxyzine-13C6, the FTC survey sample was re-analyzed and matrix effects performed.
b. To evaluate the unacceptable matrix effect study for o-desmethylvenlafaxine, the IS for the assay was reviewed. Venlafaxine-d6 is used as the IS for the quantitation of o-desmethylvenlafaxine. However, the RT differs between venlafaxine-d6 and o-desmethylvenlafaxine at 1.94 and 1.59 minutes, respectively. The matrix effect study shows an ion enhancement is occurring for o-desmethylvenlafaxine at 1.59 minutes. An isotopically matched IS o-desmethylvenlafazine-d6 was purchased from Cerilliant Corporation. The matrix effect study was repeated utilizing the MassLynx software.

4. Outcome
Here we show two cases which highlight the importance of using an isotopically matched IS in liquid chromatography-mass spectrometry (LC/MS/MS)-based assays to overcome matrix effects attributed to the sample. The IS initially used for the quantitation of hydroxyzine and o-desmethylvenlafaxine were acetopromazine and venlafaxine-d6, respectively. These IS eluted at different retention times than hydroxyzine and o-desmethylvenlafaxine. As a result the isotopically matched IS, hydroxyzine-13C6 and o-desmethylvenlafaxine-d6, were implemented and the samples were re-analyzed. The FTC survey sample for hydroxyzine changed from a +2.8 SDI to a zero SDI as compared with our peer group. Compensation for matrix effects was evident as the IS normalized factor was 1.0 with a 0.53% CV. After changing to o-desmethylvenfaxine-d6 as the IS for the measurement of o-desmethylvenlafaxine, matrix effects were repeated, and results met our acceptance criteria with a IS normalized factor of 1.08 and a 14.94% CV. The encountered matrix effect issue was resolved. In conclusion, changing the IS for both hydroxyzine and o-desmethylvenlafaxine to their isotopically matched IS, hydroxyzine-13C6 and o-desmethylvenlafaxine-d6, emphasized the importance of using isotopically matched IS in LC/MS/MS-based assays to minimize matrix effects and prevent inaccurate drug measurements.