= Emerging. More than 5 years before clinical availability. (17.55%, 2019 US)
= Expected to be clinically available in 1 to 4 years. (42.72%, 2019 US)
= Clinically available now. (39.74%, 2019 US)

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MSACL 2019 US : Truong

MSACL 2019 US Abstract


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Topic: Troubleshooting

On the Path to Standardized Approach of Matrix Effect, Process Efficiency and Recovery Assessment in Clinical Mass Spectrometry Testing

Dorothy Truong (Presenter)
LifeLabs

Presenter Bio: Dr. Dorothy Truong completed and graduated from the University of Toronto Clinical Chemistry Fellowship program. During her fellowship program, she received training in routine chemistry, mass spectrometry, therapeutic drug monitoring, toxicology and clinical immunology. Dr. Truong completed her certification requirements, and is a Fellow of the Canadian Academy of Clinical Biochemistry.

Authors: Dorothy Truong, Theano Karakosta, Dawn-Marie Murphy, Difei Sun, Danijela Konforte
LifeLabs ON, Toronto, Canada

Short Abstract

Evaluation of matrix effect is a crucial aspect of any LC-MS/MS method development and there are many published protocols describing how to test it. During method development of 17-hydroxyprogesterone, we applied a recently published protocol(1) that simultaneously assesses matrix effect, process efficiency, and recovery. Results ruled out instrumental sources of matrix effect and pointed to sample preparation as the likely source. We repeated the study following adjustments to the elution buffer until results were acceptable. We plan to utilize this protocol in future method development studies as it thoroughly assesses different sources of matrix effect and significantly reduces troubleshooting time.

Long Abstract

Problem

The matrix is defined as the components of the sample other than the analyte of interest. If the matrix is not properly controlled, a phenomenon termed, matrix effect, may lead to under- or overestimation of analyte. Since patient serum has a diverse range of matrices, matrix effects are commonly seen with these types of samples. In LC-MS/MS methods, matrix effects may impact separation performance, analyte recovery, or ionization efficiency. Methods for troubleshooting and identifying matrix effects are well-documented in the literature, with pre and post-extraction spiking experiments commonly used to identify matrix effects (2).

During method development and validation of 17-hydroxyprogesterone (17-OHP) LC-MS/MS assay, we observed poor 17-OHP internal standard recovery in patient samples relative to calibrators and quality control material. Furthermore, recovery experiments with 17-OHP standards demonstrated a positive bias between observed and expected results. While our experiments identified a matrix effect, it was insufficient in shedding light on where our troubleshooting efforts should be focused.

Bienvenu JF et al.(1), published an approach to assess matrix effect as it relates to calibration curve, sample preparation, process efficiency and instrumental matrix effect. This approach provided a complete picture of the matrix effect and gave insight on where to focus our efforts to minimize the influence of matrix effects on our analyte.

Method Information

Assessment of matrix effect was performed by following the approach published in Bienvenu JF, et al.(1)

Briefly, each specimen was prepared as follows:

Set A: Addition of 17-OHP standard and internal standard into neat reconstitution solvent

Set B: Sample preparation performed on specimen. 17-OHP standard and internal standard was added afterwards

Set C: Addition of 17-OHP standard to the specimen prior to sample preparation, and addition of internal standard afterwards.

Set D: Addition of 17-OHP standard and internal standard prior to sample preparation.

Set E: Addition of 17-OHP internal standard prior to sample preparation and no addition of standard.

Set F: Sample preparation performed on specimen, and addition of only 17-OHP internal standard afterwards.

5 different serum pools representing different types of serum commonly observed in a clinical lab: 1 lipemic specimen pool, 1 icteric specimen pool, 1 hemolyzed specimen pool, 1 specimen from a patient with a monoclonal gammopathy, and 1 pool of patient samples without interferences, were prepared as described above. 17-OHP standard was added into specimens at a concentration of 5µg/L.

The equations and terminology used in our study are defined as:

Global matrix effect: (Set D – Set E)/added concentration

Instrument Matrix effect on Concentration: (Set B – Set F)/added concentration

Internal Standard Normalized Standard Recovery Factor (bias between recovery of analyte and internal standard): (Set D – Set E)/(Set B – Set F)

Standard Recovery: (Set C – Set F)/(Set B – Set F)

Troubleshooting Steps

Using the approach published by Bienvenu JF, et al.(1), we identified a global matrix effect of 14%, indicating that on average, analyte concentration was overestimated by approximately 14%. These results are consistent with our recovery experiments demonstrating a positive bias between observed and expected results. The source of matrix effect was further broken down into instrumental and sample preparation factors. Analysis of appropriateness of calibration curve, process efficiency, internal standard recovery, and standard recovery helped deduce whether the source of matrix effect was largely due to instrumental or sample preparation sources.

We identified that instrumental matrix effect and appropriateness of calibration curve were not contributing factors to the observed matrix effect. Since instrumental matrix effect was not a contributing factor, this suggested that ionization efficiency was not affecting our analysis, and thus, the instrumental side could be eliminated as a contributing factor to the observed matrix effect.

After eliminating instrumental factors as a source of the matrix effect, we focused on sample preparation as the contributing factor to the observed matrix effect. Using the equations derived by Bienvenu JF, et al. (1), we calculated a bias of approximately 21.5% between recovery of the 17-OHP standard and 17-OHP internal standard. Furthermore, precision of standard recovery was 15% across different matrices, suggesting poor reproducibility of analyte recovery. Overall, our studies suggested that our sample preparation was sub-optimal and led to differential recovery of 17-OHP analyte and 17-OHP internal standard.

To optimize sample preparation, we increased the concentration of organic solvent in the elution buffer and replaced 100% Ethyl Acetate with a mixture of 80% Ethyl Acetate: 20% Hexane, and subsequently repeated the matrix effect study.

Optimization of our extraction buffer resulted in a global matrix effect of 0.1%. There was no change in the instrumental matrix effect and the appropriateness of the calibration curve. The bias between recovery of the 17-OHP standard and 17-OHP internal standard significantly decreased to 6.3%. Lastly, the reproducibility of analyte recovery significantly improved to 3.4%.

Outcome

Identifying and troubleshooting matrix effects can be time consuming. Our results indicate that the protocol published by Bienvenu JF et al. (1) provides a detailed overview of matrix effect, recovery, process efficiency and internal standard association. More importantly, with one experiment, scientists can identify steps in the method which require optimization, allowing for more focused studies and minimizing the amount of time spent on troubleshooting matrix effects. We plan to implement it in all future method development projects.


References & Acknowledgements:

1. Bienvenu J.F., Provencher G., Bélanger P., Bérubé R., Dumas P., Gagné S., Gaudreau E., Fleury N., Standardized Procedure for the Simultaneous Determination of the Matrix Effect, Recovery, Process Efficiency, and Internal Standard Association. Anal Chem. 2017, 89:7560-68

2. Matuszweski B. K., Constanzer M. L., Chavez-Eng C. M., Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 2003, 75:3019-3030


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