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Evaluation of LCMSMS Deuterium Scrambling in Clinically Significant Small Molecules

By: Joshua Cooper, IsilDilek, and UmaSreenivasan, Cerilliant Corporation, 811 Paloma Drive, Suite A, Round Rock, TX 78665

Introduction

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.

Comparisons of 25-Hydroxyvitamin D Deuterium Scrambling

25-Hydroxyvitamin D2                        25-Hydroxyvitamin D2-d3                    25-Hydroxyvitamin D2-d6

   

 

                        25-Hydroxyvitamin D3                            25-Hydroxyvitamin D3-d6

                   

 

Labeled 25-Hydroxyvitamin D2 and D3 Scrambling in Serum

 

Compound Label System Concentration μg/mL Transition
dn-1
Transition
dn
Scrambling %
dn-1/ dn
25-Hydroxyvitamin D2
d3
Xevo G2
2 398→379 398→380 28.6
0.2 398→379 398→380 35.4
6410
5
416→397 416→398 2.8
416→379 416→380 19.7
398→379 398→380 30.4
50
416→397 416→398 2.8
416→379 416→380 20
398→379 398→380 30.5
d6
6410
5
419→400 419→401 2
419→382 419→383 8.8
401→382 401→383 5.9
50
419→400 419→401 2
419→382 419→383 9
401→382 401→383 5.4
25-Hydroxyvitamin D3
d6
6410
2.5
407→388 407→389 4
407→370 407→371 18.8
389→370 389→371 9.2

 

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

Parent →Water loss
Compound Label Concentration μg/mL Transition dn-1 Transition dn Scrambling % dn-1/dn
25-Hydroxyvitamin D2
d3 100 416→397 416→398 2.9
d6 100 419→400 419→401 2
native 50 413→394 413→395 0.5
25-Hydroxyvitamin D3
d6 50 407→388 407→389 4
native 100 401→382 401→383 0.5
 
Parent →2 Water losses
Compound Label Concentration
μg/mL
Transition dn-1 Transition dn Scrambling % dn-1/ dn
25-Hydroxyvitamin D2
d3 100 416→379 416→380 19.5
d6 100 419→382 419→383 8.9
native 50 413→376 413→377 0.5
25-Hydroxyvitamin D3
d6 50 407→370 407→371 18.9
native 100 401→364 401→365 0.3
 
Water Loss →2 Water losses
Compound Label Concentration
μg/mL
Transition dn-1 Transition dn Scrambling % dn-1/dn
25-Hydroxyvitamin D2 d3 100 398→379 398→380 30.4
d6 100 401→382 401→383 5.4
native 50 398→376 398→377 0.4
25-Hydroxyvitamin D3 d6 50 389→370 389→371 11.2
native 100 383→364 383→365 0.3

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

                    Testosterone                                Testosterone-13C3                           Testosterone-d3

            

Major transitions are:
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

Label Method Instrument Concentration
μg/mL
Transitions
Dn-1or 13Cn-1
Transitions
Dn or 13Cn
*Scrambling %
Dn-1/ Dn
d3
Infusion
Q-Tof
10 292→255
292→256
31.9

LC

100 36.5
10 35.7

6410
100 37.7
10 36.3
13C3 100 0.1
native 100 289→252 289→253 0.0
* or Scrambling % 13Cn-1/ 13Cn

 

Testosterone Chromatograms on XevoG2

Testosterone Chromatograms on XevoG2

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

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

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

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

Everolimus Scrambling at m/z 389

Everolimus Scrambling at m/z 389

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

Compound Label Polarity Collision Energy Transition(s) dn Scrambling
% dn-1/dn
(-)-Δ9-THC d3 pos 25 318→262,196 0
native pos 25 315→259,193 0
(-)-Δ9-THC d3 neg 30 318→262,196 can't determine
native neg 30 315→259,193 can't determine
(±)-11-Hydroxy-Δ9-THC d3 pos 15 334→316 3.12
native pos 15 331→313 0
(±)-11-Hydroxy-Δ9-THC d3 pos 25 334→any can't determine
native pos 25 331→any can't determine
(±)-11-nor-9-Carboxy-Δ9-THC d9 neg 30 352→254 0
d3 neg 30 346→248 0
native neg 30 343→245 0
(±)-11-nor-9-Carboxy-Δ9-THC d9 neg 20 352→334 0
d3 neg 20 346→328 0
native neg 20 343→325 0
(±)-11-nor-9-Carboxy-Δ9-THC d9 pos 20 354→336 51.91
d3 pos 20 348→330 0
native pos 20 345→327 0
(±)-11-nor-9-Carboxy-Δ9-THC d9 pos 20 354→308 48.88
d3 pos 20 348→302 0
native pos 20 345→299 0
Cannabinol d3 neg 30 316→248 0
native neg 30 313→245 0
Cannabinol d3 pos 20 318→262,196 0
native pos 20 315→259,193 0
JWH-018 4-Hydroxypentyl metabolite d5 pos 20 363→345 22.83
native pos 20 358→340 0
JWH-018 4-Hydroxypentyl metabolite d5 pos 20 363→155 0
native pos 20 358→155 0
JWH-018 4-Hydroxypentyl metabolite d5 pos 20 349→221 25.44
native pos 20 344→216 0
JWH-073 3-Hydroxybutyl metabolite d5 pos 20 363→155 0
native pos 20 358→155 0
3,4-MDPV HCl d8 pos 15 284→134 0
native pos 15 284→126 0
Ethylone HCl d5 pos 15 227→209 0
native pos 15 222→204 0
Butylone HCl d3 pos 15 225→209, etc 0
native pos 15 222→204, etc 0
MephedroneHCl d3 pos 10 181→163 0
native pos 10 178→160 0
Methylone HCl d3 pos 10 211→163 0
native pos 10 208→160 0
Methylone HCl d3 pos 10 211→135 0
native pos 10 208→132 0
Everolimus d4 pos 60 984→393 1.80
native pos 60 980→389 0
Mycophenolic acid d3 pos 15 322→278 0
native pos 15 319→275 0

 

Conclusions

  • 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.

Materials

     
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