Evaluation of LC-MS/MS Scrambling Ratios for Deuterium-Labeled Vitamin D Metabolites, Steroids and Other Compounds of Clinical Significance

By: Joshua Cooper, Huahua Jian, Derrell Johnson, Isil Dilek, and Uma Sreenivasan, Cerilliant Corporation, 811 Paloma Drive, Suite A, Round Rock, TX 78665

Abstract

Introduction and Objective: A significant clinical challenge with LC-MS/MS is the potential for matrix effects that cause interferences or impact ionization efficiency. Stable isotope-labeled internal standards are frequently used to compensate for matrix effects and to increase the accuracy of quantitation. The use of a labeled internal standard that co-elutes with the drug being monitored can potentially offset patient specific matrix effects (co-eluting concomitant medication, etc.) that may occur at the retention time of the analyte of interest. Complications in the use of deuterium-labeled internal standards can arise from hydrogen-deuterium scrambling in the collision cell at the selected transitions or in the ion source. In this study, we examined deuterium labeled 25-Hydroxyvitamin D, testosterone, and other compounds of clinical significance by LC-MS/MS at multiple transitions. We investigated reproducibility of the scrambling ratio and influences on scrambling of different LC-MS systems (tandem quadrupole vs. quadrupole time-of-flight), matrix selection, concentration, and deuterium placement in the internal standard.

Methods and Procedures

LCMS System 1:
Instrument: Waters Alliance UPLC-Xevo G2 Q-Tof
Column: Waters Acquity UPLC, BEH C18, 1.7μm, 2.1 x 50mm

25-Hydroxyvitamin D Analysis Conditions:
UPLC Conditions: 0.4mL/min, gradient, 0.1:99.9 to 99.9:01
(0.1% formic acid in acetonitrile:0.1% formic acid in water)
MS Conditions: ESI+, Cone 25V, Capillary 2.5kV, CE 20

Testosterone Analysis Conditions:
UPLC Conditions: 0.4mL/min, isocratic, 30:70 (0.1% formic acid in acetonitrile:0.1% formic acid in water)
MS Conditions: ESI+, Cone 30V, Capillary 3.0kV, CE 18

LCMS System 2:
Instrument: Agilent 1100 HPLC-6410 triple quad
Column: Phenomenex Kinetex, C18, 3μm, 2.1 x 50mm

25-Hydroxyvitamin D Analysis Conditions:
HPLC Conditions: 0.4mL/min, isocratic, 80:20 (0.1% formic acid in methanol:0.1% formic acid in water)
MS Conditions: ESI+, Fragmentor 110V, Capillary 4.0kV, CE 5

Testosterone Analysis Conditions:
UPLC Conditions: 0.4mL/min, isocratic, 30:70 (0.1% formic acid in acetonitrile:0.1% formic acid in water)
MS Conditions: ESI+, Fragmentor 50V, Capillary 4.0kV, CE 10

Solution Standards Used:

25-Hydroxyvitamin D3, Cat# H083

25-Hydroxyvitamin D3-d6, Cat# H074

25-Hydroxyvitamin D2, Cat# H073

Testosterone, Cat# T037

Testosterone-d3, Cat# T046

Testosterone-13C3, Cat# T037

Progesterone-d9, Cat# P070

Pregabalin-d6, Cat# P072

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


Comparisons of 25-Hydroxyvitamin D2 and D3 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

 

Vitamin D in Serum on 6410

Vitamin D in Serum on 6410

EtOH Scrambling

Vitamin D in EtOH Scrambling on Xevo G2

Investigation of Method, Instrument, and Concentration Effects on Scrambling for Vitamin D

Compound Method Instrument Concentration
μg/mL
Transition dn-1 Transition dn Scrambling % dn-1/ dn
d3 labeled 25-Hydroxyvitamin D2
Infusion
Q-Tof
10 398→379
398→380
29.7
5 30.9
10 27.1
LC 6410 100 30.4
33 30.2

 

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

 

5μg/mL Infusion at 20μL/min of d3 labeled 25-Hydroxyvitamin D2 on Xevo G2

Transition
dn-1
Transition
dn
Scrambling %
dn-1/ dn
416→397 416→398 2.2
416→379 416→380 16.9
398→379 398→380 30.9

Note: Under optimized UPLC-Q-Tof conditions only water loss MS ions were detected. MS ion ratios changed for 25-Hydroxyvitam D when combined with mobile phase. Could detect ions without water loss when infusing.

Investigation of Testosterone Scrambling

                Testosterone                            Testosterone-13C3                        Testosterone-d3

      
  

 

Testosterone Chromatograms on 6410

Testosterone Chromatograms on 6410

Testosterone Chromatograms on Xevo G2

Testosterone Chromatograms on Xevo G2

Testosterone Scrambling Comparison

Label Method Instrument Concentration
μg/mL
Transitions
Dn-1or 13Cn-1
Transitions
Dnor 13Cn
*Scrambling %
Dn-1/ Dn
d3 Infusion Q-Tof
10     31.9
LC
100     36.5
10     35.7
6410
100 292→255 292→256 37.7
10     36.3
13C3 100     0.1
native 100 289→252 289→253 0.0
* or Scrambling % 13Cn-1/ 13Cn

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 m/z 253

Testosterone Scrambling at m/z 253

Testosterone Scrambling at 97 and 109

Testosterone Scrambling at 97 and 109

Testosterone dn-2/ dn Scrambling

Label Method Instrument Concentration
μg/mL
Transition
dn-2
Transition
dn
Scrambling %
dn-2/ dn
d3 Infusion Q-Tof 10 292→254 292→256 2.6
d3 LC Q-Tof 100 292→254 292→256 3.6
d3 LC Q-Tof 10 292→254 292→256 <LOD

 

Scrambling for other clinical compounds

Xevo G2 Scrambling Infusion Experiments

Compound Label Transition
dn-1
Transition
dn
Scrambling %
dn-1/ dn
Transition
dn-1
Progesterone
d9
324→305 324→306 20 19
324→287 324→288 77 19
324→112 324→113 0 19
324→99 324→100 0 19
 
Pregabalin
d6
166→147 166→148 0 25
166→129 166→130 0 25
166→102 166→103 12 25
166→88 166→89 40 25

 

Progesterone

             Progesterone-d9                                     Pregabalin-d6

  

 

Conclusions

  • Scrambling was observed on both the Agilent 6410 triple quadrupole and the Waters Xevo G2 Q-Tof, and in some cases was very pronounced.
  • 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.
  • It may be advisable to investigate at higher concentrations than normally analyzed to ensure that instrument sensitivity doesnot impact accuracy of scrambling determination.
  • Awareness of potential scrambling is important for proper internal standard selection. Scrambling may be mitigated or eliminated by altering instrument conditions and transition selection.
  • Deuterium-labeled internal standards are a viable option for LC-MS/MS analysis with selection of the appropriate transition. Deuterated standards 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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