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Adv Biomed Res 2023,  12:104

An interfering substance in gel tubes affects vitamin D measurement by HPLC

Reference Clinical Laboratory, Qom University of Medical Sciences, Qom, Iran

Date of Submission30-Jul-2022
Date of Acceptance09-Oct-2022
Date of Web Publication27-Apr-2023

Correspondence Address:
Dr. Mohammad Reza Haeri
Reference Laboratory, Jomhoori Street, Lane No. 2, Qom
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abr.abr_252_22

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How to cite this article:
Haeri MR, Emamnejad N. An interfering substance in gel tubes affects vitamin D measurement by HPLC. Adv Biomed Res 2023;12:104

How to cite this URL:
Haeri MR, Emamnejad N. An interfering substance in gel tubes affects vitamin D measurement by HPLC. Adv Biomed Res [serial online] 2023 [cited 2023 Jun 7];12:104. Available from:

To the Editor,

The demand for measuring vitamin D has increased dramatically, thus vitamin D measurement is one of the most frequently requested laboratory tests.[1] One of the most common methods to measure 25-hydroxy vitamin D3 (25-OH-D3) is high-performance liquid chromatography (HPLC) with enough reliability and high selectivity.[2] There is little information about the potential impact of blood collection tubes on 25(OH) D3 concentrations. However, many factors may affect the accuracy of the measurements, mainly pre-analytical variables such as sample type and interfering factors.[3] The type of blood samples (plasma or serum) or collecting tube (plain or clot-activating tube) sent from the hospital wards to the laboratory may vary, depending on the tests requested for the patient. The question of this study was whether the amounts of 25(OH) D3 measured by the HPLC method in serum (prepared in tubes containing gel and clot activator) and plasma are the same. For this purpose, blood samples from eight patients were collected simultaneously in tubes containing gel and clot activator, and in tubes containing EDTA (without gel). All tubes were centrifuged for 10 min at 3000 × g. Further, 25(OH) D3 was measured in all samples by HPLC (Agilent, USA) equipped with a C18 column and ultraviolet (UV) detector adjusted to 264 nm.[4] The mobile phase consisted of acetonitrile/methanol (90/10). To prepare samples, 400 μL of the patient sample and 400 μL of the precipitation and extraction reagents were dispensed into test tubes. To obtain a precipitate, the tubes were vortex-mixed for 10 s and centrifuged at 10,000 RCF for 5 min. Finally, 250 microliters from the supernatant were injected into the HPLC, the mobile phase was applied with a flow rate of 1 mL/min in isocratic elution mode. Results were compared with the student's t-test using the GraphPad Prism 8.2.1 software. The significance level was defined as P ≤ 0.05.

As shown in [Figure 1], the area under the chromatogram curve for serum obtained from gel-activating tubes and EDTA-plasma was different. Also, 25(OH) D3 levels in serum were significantly lower than those in the plasma (13.4 ± 11.6 vs. 16.3 ± 12.4) (P = 0.001). There are opposing pieces of evidence regarding the difference between serum and plasma vitamin D levels using immunoassay methods.[5],[6] To our knowledge, interference with HPLC has not been yet clearly reported. In our study, statistical analysis showed that 25(OH) D3 levels in serum obtained from the gel-activating tube are significantly different from plasma (P = 0.001). It seems that additives (gels and/or clot activator) may create a source of perturbation on 25(OH) D3 chromatographic movement through the column and then affect elution time. Lensmeyer et al.[7] reported a similar phenomenon; however, despite what they reported, we observed a distinct and definite extra peak when using gel tubes [Figure 1]. The exact mechanism of action is not clear, we purpose that gel and/or clot activator may interact with 25(OH) D3 to separate part of the 25(OH) D3 from the main peak, creating an extra peak; therefore, the amount of area under the curve of the main peak decreases, and measured 25(OH) D3 is reduced to less than the actual amount. We concluded that the preferred sample for measuring 25(OH) D3 by HPLC is serum or plasma without any additive such as gel or clot-activating materials.
Figure 1: Overlapping the two chromatograms obtained from EDTA-plasma and serum taken in gel tubes showing the difference in the area under the curve in two measurements, the arrow indicates the extra peak

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We sincerely thank Mr. Ebadi for providing materials and instruments.

Ethical approval

Samples were taken from patients who had come to the hospital and vitamin D measurements were part of their prescribed test list; therefore, ethical approval was waived by the Ethics Committee of Qom University of Medical Sciences, given that all procedures performed were part of the patient's routine care.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Caillet P, Goyer-Joos A, Viprey M, Schott A. Increase of vitamin D assays prescriptions and associated factors: A population-based cohort study. Sci Rep 2017;7:10361.  Back to cited text no. 1
Enko D, Fridrich L, Rezanka E, Stolba R, Ernst J, Wendler I, et al. 25-hydroxy-Vitamin D status: Limitations in comparison and clinical interpretation of serum-levels across different assay methods. Clin Lab 2014;60:1541-50.  Back to cited text no. 2
Bowen RA, Hortin GL, Csako G, Otañez OH, Remaley AT. Impact of blood collection devices on clinical chemistry assays. Clin Biochem 2010;43:4-25.  Back to cited text no. 3
Keyfi F, Nahid S, Mokhtariye A, Nayerabadi S, Alaei A, Varasteh A. Evaluation of 25-OH vitamin D by high performance liquid chromatography: Validation and comparison with electrochemiluminescence. J Anal Sci Technol 2018;9.   Back to cited text no. 4
Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochem Med (Zagreb) 2013;23: 321-5.  Back to cited text no. 5
Yu S, Zhou W, Cheng X, Fang H, Zhang R, Cheng Q, et al. Blood collection tubes and storage temperature should be evaluated when using the siemensADVIA centaur XP for measuring 25-hydroxyvitamin D. PLoS One 2016;11:e0166327.  Back to cited text no. 6
Lensmeyer GL, Wiebe DA, Binkley N, Drezner MK. HPLC method for 25-hydroxyvitamin D measurement: Comparison with contemporary assays. Clin Chem 2006;52:1120-6.  Back to cited text no. 7


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