Evaluation of LCMSMS Deuterium Scrambling in Clinically Significant Small Molecules

Joshua Cooper, IsilDilek, Uma Sreenivasan

Cerilliant Corporation, 811 Paloma Drive, Suite A, Round Rock, TX 78665

Introduction and Objective

LC-MS/MS is a powerful tool that brings numerous benefits to the clinical sample analysis arena. However, due to the complexity of the instrumentation there are some unique challenges that also accompany these benefits. Even following sample extraction and cleanup, matrix effects from the samples can cause interferences or impact ionization efficiency. Deuterium-labeled internal standards are the most common and prevalent labeled internal standards used to compensate for matrix effects. Some deuterium labeled compounds may exhibit hydrogen-deuterium scrambling/exchange in the collision cell which can impact MS/MS transition selection.

In this study we investigated numerous variables that potentially contribute to scrambling in order to ascertain reproducibility and impact on scrambling ratios: influencesof different LC-MS systems (tandem quadrupolevs. quadrupoletime-of-flight), matrix selection, concentration, with and without HPLC, collision energies, and deuterium placement in the internal standard. Numerous small molecules of clinical importance were investigate including: hydroxyvitaminD, testosterone, immunosuppressants, bath salts, and spice cannabinoids.

Materials and Procedures

LCMS Systems

  • Waters Alliance UPLC-XevoG2 Q-Tof
  • Agilent 1100 HPLC-6410 triple quad

Cerilliant Solution Standards Used

Serum Extraction

  1. 200μL of sample in serum + 200μL of methanol, vortexed to mix.
  2. Added 1mL of heptane, vortexedfor 30sec,
  3. Centrifuged for 4min at 3000rpm
  4. 900μL of top layer dried under nitrogen
  5. Reconstituted in 100μL of ethanol

Comparisons of 25-Hydroxyvitamin D Deuterium Scrambling

25-Hydroxyvitamin D2

25-Hydroxyvitamin D2

25-Hydroxyvitamin D2-d3

25-Hydroxyvitamin D2-d3

25-Hydroxyvitamin D2-d6

25-Hydroxyvitamin D2-d6

25-Hydroxyvitamin D3

25-Hydroxyvitamin D3

25-Hydroxyvitamin D3-d6

25-Hydroxyvitamin D3-d6

Labeled 25-Hydroxyvitamin D2 and D3 Scrambling in Serum

Transitions Comparisons for Native and Labeled 25-Hydroxyvitamin D2 and D3 in EtOH on 6410

Notes: 25-Hydroxy D2-d6 water loss→2 water loss has same transition as 25-Hydroxyvitamin D3 parent→waterloss. Can be problem if compounds are not well resolved chromatographically.

Selection of Transitions Greatly Impacts Observed Scrambling

Investigation of Testosterone Scrambling







Major Transitions

  • Native: 289→97 & 289→109
  • Testosterone-d3: 292→97 & 292→109
  • Testosterone-13C3: 292→100 & 292→112
  • No scrambling at major transitions

Testosterone Scrambling at Minor Transitions

* or Scrambling %13Cn-1/13Cn

Testosterone Chromatograms on XevoG2

Figure 1. Testosterone Chromatograms on XevoG2

(±)-11-nor-9-Carboxy-Δ9-THC Scrambling at m/z 327

Figure 2. (±)-11-nor-9-Carboxy-Δ9-THC Scrambling at m/z 327

JWH-073 3-Hydroxybutyl Metabolite Scrambling at m/z 216

Figure 3. JWH-073 3-Hydroxybutyl Metabolite Scrambling at m/z 216

Everolimus Scrambling at m/z 389

Figure 4. Everolimus Scrambling at m/z 389

Spice Cannabinoids, Bath Salts, and Immunosuppressants Scrambling Comparison usinf Xevo G2


  • Scrambling was observed for several of the analytesat select transitions. In all cases, scrambling was mitigated or eliminated by optimizing instrument conditions and transition selection.
  • Awareness of potential scrambling is important for proper internal standard selection.
  • Scrambling was observed on both the Agilent 6410 triple quadrupoleand the Waters XevoG2 Q-Tofto approximately the same degree. For a specific transition, scrambling ratios were consistent between solvent and serum.No matrix effects on scrambling.
  • Direct infusion can provide rapid and accurate determination of scrambling ratios.Infusion and chromatographic injection results were consistent.
  • Scrambling may be mitigated or eliminated by altering instrument conditions and transition selection. However, there is a need to consider potential impact of scrambling on transitions chosen for optimal sensitivity.
  • Deuterium-labeled internal standards are a viable option for LC-MS/MS analysis with selection of the appropriate transition. Deuteratedstandards can be more cost effective than 13C labeled internal standards, more widely available and with lower cost per test.13C labeled internal standards are most effective when deuterium scrambling issues can not be resolved.

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