13C-Labeled Internal Standards for LC-MS/MS Analysis. Huiling Liu, Morten Karlsen, Håkon Midtaune, Jon E. Johansen. Chiron AS

Transcription

1 C-Labeled Internal Standards for LC-MS/MS Analysis uiling Liu, Morten Karlsen, åkon Midtaune, Jon E. Johansen Chiron AS

2 Projects for developing Pharmaceutical and narcotic reference materials: Industrial Ph. ( ), - The Research Council of Norway - Cooperated with NTNU ISFR ( ), - Innovation Norway - Cooperated with NIP Quantum SPICE ( ), - EuroStars, European Funding - Cooperated with Nal Van Mitten

3 Internal Standards (ISs): The IS is added prior to any manipulations in the analysis procedure To adjust for loss and fraction, and thereby to improve qualitative and quantitative detection Import properties for a perfect IS: Similar or identical (ideally) chemical and physical properties as the analyte Absent in the sample etected separately from the analyte

4 IS Sample prep Chromatography Mass spectrometry If a known quantity of IS is added to the unknown sample prior to any manipulations, the ratio of IS to analyte, remains constant, because the same fraction of each is lost in any operation.

5 Where can loss and fractionation occur: uring sample preparation and instrumental analysis Ion suppression in MS detection is a major concern

6 IN SUPPRESSIN: ESI Compound of interest Matrix (better sample prep) Co-eluting compounds (better chromatography)

7 Stable Isotope Labeled Internal Standards - SIL-ISs Import properties for a perfect ISs: Similar or identical (ideally) chemical and physical properties as the analyte Absent in the sample etected separately from the analyte co-eluted chromatographically with analyte Stable Isotope labeled ISs: 2, C, 15 N, 18 Can all SIL-ISs be the perfect ISs?

8 C-labeled ISs vs. 2 -labeled ISs - Results from ISFR, Innovation Norway Project - Coopeartion with Norwegian Institute of Public ealth (NIP) C-labeled ISs are superior to deuterium labelled IS C- labeled ISs give better: Ion suppression correction Accuracy Precision

9 Amphetamines N 2 N N 2 Amphetamine Methamphetamine Phentermine N 2 N N MA MMA MEA Chiron produces native, 2 and C labeled standards

10 % C-labeled IS vs. 2 -labelled IS: UPLC-MS/MS (Thomas Berg and ag. Strand, NIP) 100 Column: Acquity BE C 18 (50 x 2.1 mm) C 3 C 2 C N 2 Amphetamine Mobile phase: A: 5mM N4-formate buffer, p 10.2 B: Me Time min

11 % UPLC-MS/MS (Thomas Berg and ag. Strand, NIP) 100 C C 2 C N 2 Amphetamine Time min

12 UPLC-MS/MS (Thomas Berg and ag. Strand, NIP) 100 C C C N 2 % Amphetamine Time min

13 UPLC-MS/MS (Thomas Berg and ag. Strand, NIP) 100 C C C C C C C 3 C 2 C N 2 C 6 -Amphetamine % Time min

14 Measured Concentration µm 1000 Amphetamine concentration 12 x 3 extracted standard samples Y = X Amph. C 6 -A A- 3 A For calculation IS=Amphetamine Spiked concentration µm

15 Measured Concentration µm 1000 Amphetamine concentration 12 x 3 extracted standard samples Y = X Amph. C 6 -A A- 3 A- 11 Amphetamine -70% ion suppression 200 IS=Amphetamine Spiked concentration µm

16 Measured Concentration µm 1000 Amphetamine concentration 12 x 3 extracted standard samples Y = X IS=Amphetamine IS=Amphetamine Spiked concentration µm

17 Measured Concentration µm 1000 Amphetamine concentration 12 x 3 extracted standard samples Y = X 800 IS= C 6 -Amphetamine 600 IS=Amphetamine IS=Amphetamine Spiked concentration µm

18 Measured Concentration µm 1000 Amphetamine concentration 12 x 3 extracted standard samples Y = X 800 IS= C 6 -Amphetamine Improved Accuracy and Precision IS=Amphetamine IS=Amphetamine µm Spiked concentration

19 C-labeled Internal Standards ( C-ISs) - The GL Standard for LC-MS/MS C-ISs are superior to euterated ISs in UPLC-MS/MS analysis of drugs The same RETENTIN TIMES and RESPNSE FACTRS as the native analytes lead to No Bias with Ion Suppression

20 C-labeled amphetamine standards:

21 ther C-ISs at Chiron: pioids: Morphine, codeine, heroine and metabolites N N N Morphine- C 1 Codeine- C 2 Ethylmorphine- C 3 N N N 3-MAM- C 3 6-MAM- C 3 eroin- C 5

22 ther C-ISs at Chiron: Cannabinoids: TC, TC-C, TC- C 4 Δ9-TC C Δ9-TC C C 4 11-C-Δ9-TC