MSACL 2023 Abstract

Determination of Free Thyroxine from Human Serum Using BioSPME Sample Preparation Prior to LC-MS/MS Analysis M. James Ross (1), Julie A Ray (2) (1) MilliporeSigma, Bellefonte, PA (2) ARUP Laboratories, Salt Lake City, UT Olga Shimelis, PhD (Presenter) MIlliporeSigma

Presenter Bio: Worked for MilliporeSigma at the Bellefonte, PA site for the past 18 years in several R&D roles. Prior to that was a Research scientist at Northeastern University, Boston, MA with focus on identification and analysis of DNA adducts. Holds a PhD in Analytical Chemistry from the University of Oklahoma.

Introduction:

Absolute free thyroxine concentrations remain relatively constant, approximately 0.02% of total thyroxine (tT4) is available in circulation in the free form. The vast majority is bound to thyroxine-binding globulin and to a lesser extent albumin and transthyretin. Direct immunoassays, the most common test performed at clinical laboratories, suffer from interferences and lack of specificity. In a 2002 study of > 5,000 patients, reported this incident occurring in ~0.5% of samples (Ismail AAA et al. 2002, Ann Clin BioChem). Liquid chromatography tandem mass spectrometry (LC/MS/MS) methods have been developed over the past 10 years to afford improved specificity and accuracy. The current standard for separation of free hormones prior to analysis has been equilibrium dialysis (ED). In general, ED is a lengthy process, on average overnight at minimum. A novel sample preparation has been utilized to alleviate this. Solid phase microextraction, SPME, has been developed into a 96-pin device termed BioSPME to prepare samples in under one-hour.

Objective:

A collaboration was established between MilliporeSigma and ARUP Laboratories to investigate the results of sample preparation by a BioSPME device or ED prior to LC/MS/MS analysis for measurement of free thyroxine in serum.

Methods:

The free thyroxine (fT4) and/or free triiodothyronine (fT3) were analyzed on Agilent 1290 LC utilizing an Ascentis Express Biphenyl (10 cm x 2.1 mm, 2.7 µm) connected to an AB Sciex 6500 QQQ. Quantifier and qualifier transitions were utilized for unlabelled and isotopically 13C6-labelled thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). The extracted calibration standards were used to directly report the concentrations of fT4 and/or fT3.
Initial method development utilized bulk serum that was tested by an external clinical laboratory for independent determination of fT4 and fT3 by a validated ED-LC-MS/MS method. These samples were additionally used to perform reproducibility assays of the method.

The BioSPME sample preparation method consisted of using 200 µL volume samples and a C18 coated 96-pin device with a Hamilton Starlet system for automation. The method included multiple steps of transferring the device between four well plates (condition, wash, sample, and desorption). The sample plate consisted of calibration standards prepared in 7.5 mM HEPES at pH 7.5 and serum samples which were diluted with 5% (v/v) of 1.15 M HEPES to adjust the pH. Extracted samples were desorbed into 40 µL of methanol containing internal standards. The samples were then diluted with 40 µL of water by the autosampler prior to injection.

Reinjection of extracted samples was investigated using 29 serum samples and 7 calibrators.

Serum samples for robustness testing were provided in conjunction with ARUP Laboratories (Salt Lake City, UT) and were previously determined by equilibrium dialysis-LC-MS/MS using the method described by Bingfang Yue (Yue, B, Rockwood, A, et al. 2008, Clinical Chem).

Results:

Six commercial serums were tested on five (n=4 per extraction) separate occasions to test reproducibility of the method for fT4 and fT3. On average, the interday %CV for fT4/fT3 were as follows: 10.9/9.2%, 5.5/9.2%, 8.6/6.9%, 10.2/9.5,% 4.0/8.5%, and 7.8/7.2%.

A series of different sets of serum samples were investigated. The first set of 24 (n=3), a correlation of ED-LC-MS/MS to BioSPME-LC-MS/MS yielded a relationship of y = 0.738x + 0.322, R2 = 0.867. The average % CV for the fT4 concentration was 8%. On average, fT4 values for BioSPME-LC-MS/MS were 3% lower than ED-LC-MS/MS.

In a second set of 45 samples (n=1), the overall correlation was y = 0.781x + 0.464, R2 = 0.819. On average, the fT4 values using BioSPME-LC-MS/MS were 22% lower than ED-LC-MS/MS. Additional method development is on-going to increase the accuracy of the BioSPME-LC-MS/MS method.

A study involving 29 serum samples and 7 calibrators were reinjected non-sequentially twice. On average, there was a 4.1% difference in concentration.

The LLOQ of the extracted calibrators was 1 pg/mL with a 40 µL injection with an % CV of 10.9% and 19.8% (quantifier and qualifier). The peak integration ratio for the LLOQ for the quantifier/qualifier was 1.00, % CV 14.6%. The sensitivity was achieved by replacing the 20 µL standard injection loop on Agilent 1290 LC instrument with a 100 µL loop to allow for larger injection volumes.

Conclusion:

A BioSPME extraction method prior to analysis by LC-MS/MS was developed, and the evaluation results showed strong correlation against equilibrium dialysis for determination of free thyroxine (fT4) from serum samples. The BioSPME method was automated by using a Hamilton robotic system and can be adapted to other robotic liquid handlers that have gripper functionality. The time to process one 96-well plate was less than an hour. The developed LC-MS/MS detection method included a built-in preconcentrated sample (5x) without dry-down steps and the ability of get repeat injections with consistent results.