Impact of Blood Collection Materials on LC-MS/MS Measurement of Vitamin D

While liquid chromatography tandem mass spectrometry (LC-MS/MS) has become widely recognized as the method of choice for the measurement of drugs in blood, the usage of this technique in various clinical laboratories faces challenges related to the preanalytical process. In a new study published in The Journal of Applied Laboratory Medicine (1), researchers investigated how the materials that are used in blood collection tubes influence the measurement of serum 25-hydroxyvitamin D [25(OH)D] by LC-MS/MS.

Although LC-MS/MS has become the gold standard for analysis of some steroidal hormones and drugs, over the past decade, implementation of the process in various 24/7 clinical laboratories all over the world is still a challenging endeavor (2,3). One major challenge is the need for variations in the preparation and analytical process that differ from those of traditional laboratory testing. Sample preparation methods, for example, including elution conditions, and data processing approaches, such as batch mode processing and postanalytical processing, can differ depending on the laboratory (4). Even when a system demonstrates an adequate level of efficacy, there are still major measurement errors that may arise during the steps prior to LC-MS/MS.

Recent studies have shown that substances eluted from blood collection tubes may cause noticeable errors in measurement, specifically noting interference by sodium fluoride in testosterone measurement, the adsorption of drugs targeted by the administration of the technique by separator gels, and the impact of ondansetron intake on the measurement of levels of vitamin D (5-7). Some of the variables that are required for performance evaluation and validation of LC-MS/MS differ from those in the validation of traditional testing methods, such as enzyme-linked immunosorbent assay (ELISA). It is important to identify the components and preanalytical processes that can interfere with the measurement results (1).

In this study, serum samples were collected in 20 different blood collection tubes made by various manufacturers. The samples were then processed under controlled conditions to simulate clinical practice, followed by measurement using an LC-MS/MS system. The data were compared with measurements which were obtained from a chemiluminescent enzyme immunoassay. Noteworthy variability in measurement outcomes was noted depending on the blood collection tube type that was used. Specific blood collection tubes from a manufacturer that employed specific separator gels exhibited interference, as indicated by broad peaks in chromatograms, which complicated the quantification of 25(OH)D. This interference was consistent across numerous samples, which, to the researchers, revealed a systemic problem related to the materials that were used in the blood collection tubes (1).

The findings illustrate the need for the evaluation of blood collection tubes from various manufacturers before the clinical implementation of LC-MS/MS measurements. Due to LC-MS/MS having unique potential error sources, the results, highlight the importance in utilizing LC-MS/MS international standards for insuring accurate and reliable results in clinical laboratories (1).

References

1. Ochi, S.; Kunisawa, A.; Matsuura, T. et al. The Preanalytical Process Matters: Impact of Blood Collection Tubes on the Measurement of Vitamin D Using Liquid Chromatography Tandem Mass Spectrometry. J. Appl. Lab. Med. 2025, jfaf141. DOI: 10.1093/jalm/jfaf141
2. Seger, C.; Salzmann, L. After Another Decade: LC-MS/MS Became Routine in Clinical Diagnostics. Clin. Biochem. 2020, 82, 2-11. DOI: 10.1016/j.clinbiochem.2020.03.004
3. Casals, G.; Costa, R. F.; Rull, E. U. et al. Recommendations for the Measurement of Sexual Steroids in Clinical Practice. A Position Statement of SEQC^ML/SEEN/SEEP. Adv. Lab. Med. 2023, 4 (1), 52-69. DOI: 10.1515/almed-2023-0020
4. Greaves, R. F. LC-MS/MS Random Access Automation - A Game Changer for the 24/7 Clinical Laboratory. Clin. Chem. Lab. Med. 2024, 62 (7), 1249-1251. DOI: 10.1515/cclm-2024-0501
5. Ceponis, J.; Swerdloff, R.; Leung, A. et al. Accurate Measurement of Androgen after Androgen Esters: Problems Created by ex vivo Esterase Effects and LC-MS/MS Interference. Andrology 2019, 7 (1), 42-52. DOI: 10.1111/andr.12554
6. Schrapp, A.; Mory, C.; Duflot, T. et al. The Right Blood Collection Tube for Therapeutic Drug Monitoring and Toxicology Screening Procedures: Standard Tubes, Gel or Mechanical Separator? Clin. Chim. Acta 2019, 488, 196-201. DOI: 10.1016/j.cca.2018.10.043
7. Fortuna, D.; Korn, W. R.; Brune, M. J. et al. Observation of a Positive Interference in LC-MS/MS Measurement of d₆-25-OH-vitamin D3. Clin. Mass Spectrom. 2017, 3, 22-24. DOI: 10.1016/j.clinms.2017.06.001