The Becton Dickinson Barricor TM Tube Does Not Introduce Isobaric Interferences in Mass Spectrometry-based Measurements of Testosterone

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1 The Becton Dickinson Barricor TM Tube Does Not Introduce Isobaric Interferences in Mass Spectrometry-based Measurements of Testosterone Chase Mazur 1 and Joshua Hayden 1 * Background: Testosterone analysis by mass spectrometry is essential for accurate low-level results. Unfortunately, the commonly used gel-based separator tubes introduce an intense, isobaric interference that can coelute with testosterone and impede analysis. The new Becton Dickinson (BD) 2 Barricor TM tube (BT) uses an elastomer separator that might not introduce such interferences. Methods: Testosterone concentrations and chromatograms were obtained for plasma samples from plasma separator tubes (PSTs) and the new BTs using a laboratory-developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results: Intense signals close to the testosterone retention time were observed in the testosterone quantifier transition ( ) from the PSTs; resolving these signals required longer chromatographic run times. No isobaric interferences were introduced by the BTs, allowing for shorter chromatographic run times and faster inject-to-inject times. Conclusion: BTs do not introduce the interferences observed in PSTs in LC-MS/MS testosterone analysis. These tubes can be used as a single tube that both separates cells and is amenable to LC-MS/MS testosterone analysis without additional chromatographic run times. IMPACT STATEMENT Gel-based separator tubes introduce intense, isobaric interferences in LC-MS/MS testosterone assays. These interferences require longer chromatographic run times/longer inject-to-inject times or drawing of an additional nongel tube; neither is an ideal solution. This work shows that the new BD Barricor TM tube does not introduce these interferences, and so a single cell separator tube can be drawn that is amenable to subsequent LC-MS/MS testosterone analysis. 1 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY. *Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 1300 York Ave., Box 79, New York, NY Fax (212) ; jah9108@med.cornell.edu. DOI: /jalm American Association for Clinical Chemistry 2 Nonstandard abbreviations: BD, Becton Dickinson; BT, Barricor TM tube; PST, plasma separator tube; LC-MS/MS, liquid chromatography tandem mass spectrometry; SST, serum separator tube; MTBE, methyl tert-butyl ether. May : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

2 Barricor Tube for LC-MS/MS Testosterone Analysis Gel-based cell separator tubes are the most commonly used blood collection tubes in clinical chemistry laboratories owing to the numerous advantages they offer improved stability of several analytes, easier sampling from the primary tube, reduced/easier aliquoting, and higher yields of usable serum or plasma (1). However, the thixotropic polymeric gels are composed of an array of chemicals (viscous liquids, organic and inorganic fillers, tackifiers) that can interfere with sample analysis (2). One well-reported example of this is the introduction of a closely eluting interference isobaric with the common testosterone quantifier transition (289 m/z 97 m/z) used in liquid chromatography tandem mass spectrometry (LC-MS/MS) (3 5). This interference is especially problematic in samples with low testosterone levels, which is exactly when LC-MS/MS measurements are especially important (6, 7). Chromatographic resolution of the interference requires longer chromatographic run times (reducing the number of samples that can be processed). Many laboratories therefore request an additional nongel tube for the testosterone analysis, increasing the amount of blood drawn and potentially contributing to iatrogenic anemia with its associated negative outcomes (8). Ideally, laboratories would be able to collect a single tube that separates cells from plasma and does not contribute interferences in LC-MS/MS testosterone assays. Becton Dickinson (BD) recently released the Barricor TM tube (BT) as a new blood collection tube that contains an elastomer that mechanically separates the plasma from the cells. Because this tube does not use a gel-based separator, the aim of this study was to see if it introduced any interferences in subsequent LC-MS/MS testosterone analysis. METHODS Remnant plasma from paired samples collected in PSTs and BTs were used for this study; these samples had been collected as part of a larger validation of the BT in our laboratory. The plasma used here had been stored at 4 C for >72 h before testosterone analysis. Remnant samples collected in serum separator tubes (SSTs) were also analyzed. For analysis, 100 μl of sample was mixed with testosterone-2,3,4-13 C 3 as an internal standard (1 ng/dl final concentration) and allowed to equilibrate for 10 min. A liquid liquid extraction was then performed by adding 300 μl of methyl tert-butyl ether (MTBE), vortex mixing for 60 s, and letting the samples sit for 30 min. After centrifugation (10000g for 4 min), 150 μl of the supernatant was aspirated into a new tube, where it was evaporated to dryness with air at room temperature, reconstituted with 50 μl of 30% methanol:water, and transferred to LC-MS autosampler vials. Samples (2 μl) were then injected onto an Agilent 1290 Infinity II LC system with an Agilent Poroshell 120 C18 ( mm, 2.7 μm) column heated to 50 C. The mobile phases used were A: 5 mm ammonium formate in water and B: 5 mm ammonium formate in methanol. The gradient runs from initial conditions of 30% B to 70% B at 6.50 min, followed by a wash with 100% B for 1 min, aqueous equilibration at 10% B for 1 min, and re-equilibration with the starting conditions (30% B) for 1 min ahead of the following injection. Overall inject-to-inject time for this method was 9.5 min. Analysis was performed in positive ion mode on an Agilent 6495 ion funnel mass spectrometer with a quantifier ion transition ( , collision energy 25 V) and qualifier ion transition ( , collision energy 29 V). The dwell time was 100 ms for all transitions. The capillary voltage was 3000 V with an 11 L/min flow of 400 C nitrogen sheath gas. The nitrogen source gas flow was 15 L/min with a temperature of 200 C. Ultrahigh purity nitrogen was used for the collision cell. RESULTS AND DISCUSSION Consistent with published reports (3 5), intense interference in the testosterone quantifier ion transition ( ) was observed when analyzing plasma from PSTs. A representative example 2 JALM :06 May 2018

3 Barricor Tube for LC-MS/MS Testosterone Analysis FOCUSED REPORT Fig. 1. Extracted ion chromatogram for the testosterone quantifier transition ( ) for a representative plasma sample collected in a PST. Testosterone (*) elutes at 5.1 min and was quantified at 20 ng/dl. The intense peaks noted at 3.3, 4.2, and 5.6 min were identified as isobaric interferences introduced by the PST. May : JALM 3

4 Barricor Tube for LC-MS/MS Testosterone Analysis Fig. 2. Extracted ion chromatogram for the testosterone quantifier transition ( ) for a representative plasma sample collected in a BT. Testosterone (*) elutes at 5.1 min and was quantified at 20 ng/dl. is shown in Fig. 1; comparable interference peaks were found in the 9 PSTs tested and in the SSTs tested (data not shown). These interferences showed substantial intensity, especially in comparison to lower levels of testosterone (20 ng/dl in the sample shown). Although these interferences were well separated from the testosterone peak, this separation required a longer chromatographic run time. 4 JALM :06 May 2018

5 Barricor Tube for LC-MS/MS Testosterone Analysis FOCUSED REPORT In contrast to plasma from PSTs, the extracted ion chromatograms of samples collected in BTs showed no interferences in the testosterone quantifier ion transition (Fig. 2). Of note, the testosterone peak has comparable intensity ( cps) in both chromatograms (Figs. 1 and 2). The peak appears substantially smaller in Fig. 1 because the y-axis was scaled for the intense interferences from the PST that dwarf the testosterone peak. In the absence of interferences from the PSTs, the chromatographic run time could be shortened to allow shorter inject-to-inject times as long as adequate resolution of the isobaric testosterone interferences dehydroepiandrosterone and epitestosterone was maintained. This was achieved in our method using a gradient that ran from initial conditions of 40% B to 70% B at 5.0 min. This reduced the run time by 1.5 min per sample, thus saving almost 2.5 h for a 96-well plate. However, it should be noted that optimization of the original chromatography could have been more thoroughly investigated and thus the time savings is an approximation. In conclusion, BTs overcome the long-standing problem gel tubes (PSTs and SSTs) have with regard to LC-MS/MS analysis of testosterone, namely, the introduction of intense isobaric interferences that can impede analysis. Although historically this interference has been mitigated through collection of a nongel tube (potentially contributing to iatrogenic anemia) or extended chromatography (decreasing sample throughput), the BTs offer a more advantageous solution. These tubes allow for collection of a single cell separator tube that does not introduce interferences into samples when analyzed for testosterone by LC- MS/MS. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: C. Mazur, Weill Cornell Medicine. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: J. Hayden, Becton Dickinson. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: No sponsor was declared. REFERENCES 1. Bowen R, Hortin G, Csako G, Otañez O, Remaley A. Impact of blood collection devices on clinical chemistry assays. Clin Biochem 2010;43: Bowen R, Remaley A. Interferences from blood collection tube components on clinical chemistry assays. Biochem Medica 2014;24: Shi R, van Rossum H, Bowen R. Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: interference from blood collection tubes. Clin Biochem 2012;45: French D. Development and validation of a serum total testosterone liquid chromatography tandem mass spectrometry (LC MS/MS) assay calibrated to NIST SRM 971. Clin Chim Acta 2013;415: Wang C, Shiraishi S, Leung A, Baravarian S, Hull S, Goh V, et al. Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: interference and comparison with established methods. Steroids 2008;73: Herold DA, Fitzgerald RL. Immunoassays for testosterone in women: better than a guess? Clin Chem 2003;49: Kushnir M, Blamires T, Rockwood A, Roberts W, Yue B, Erdogan E, et al. Liquid chromatography tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals. Clin Chem 2010; 56: Thavendiranathan P, Bagai A, Ebidia A, Detsky A, Choudhry N. Do blood tests cause anemia in hospitalized patients? J Gen Intern Med 2005; May : JALM 5