© 2012 MicroConstants, Inc. All Rights Reserved.
CACO-PBSS Mini-Symposium:
Bioanalytical and Analytical Applications
and P...
© 2012 MicroConstants, Inc. All Rights Reserved.
Background & Introduction
Gentamicin is an aminoglycoside antimicrobial a...
© 2012 MicroConstants, Inc. All Rights Reserved.
The Analytical Challenge
• Gentamicin is associated with severe side effe...
© 2012 MicroConstants, Inc. All Rights Reserved.
Approaches in the Literature
• USP monograph. Pre-column labeling with o-...
© 2012 MicroConstants, Inc. All Rights Reserved.
Our Approach
• Common themes: Ion Pairing or Derivatization
• No preceden...
© 2012 MicroConstants, Inc. All Rights Reserved.
Initial Results
• Made
considerable
progress
• Sensitivity
looked
promisi...
© 2012 MicroConstants, Inc. All Rights Reserved.
Results Over Time
• Vancomycin was
successfully validated
using an analog...
© 2012 MicroConstants, Inc. All Rights Reserved.
How About Derivatization with LC/MS/MS?
1. Needs to tag all 5 amines effi...
© 2012 MicroConstants, Inc. All Rights Reserved.
We Gave it a Try…
• Given the attributes of a good derivative (previous s...
© 2012 MicroConstants, Inc. All Rights Reserved.
Gentamicin Form: C1a C2 & C2a C1
Original Parent 450 464 478
Derivative (...
© 2012 MicroConstants, Inc. All Rights Reserved.
Proof of Concept… Does it Work?
• Tested extraction of 50
µL of plasma an...
© 2012 MicroConstants, Inc. All Rights Reserved.
The Solution
• Stable labeled I.S. is
synthesized by pre-
derivatization ...
© 2012 MicroConstants, Inc. All Rights Reserved.
Final Outcome
Note: Each isoform has its own I.S in the same relative abu...
© 2012 MicroConstants, Inc. All Rights Reserved.
Validation Summary
Gentamicin C1 Concentration
(ng/mL)
Gentamicin C1a Con...
© 2012 MicroConstants, Inc. All Rights Reserved.
Summary & Conclusions
• Novel application of deuterium labeled derivatiza...
© 2012 MicroConstants, Inc. All Rights Reserved.
Acknowledgements
Scientific support
• Noel Henderson
• David (Del) Lewist...
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A Novel Approach to Internal Standardization in LC/MS/MS Analysis; Sensitive LC/MS/MS Analysis of Gentamicin | MicroConstants

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This presentation was prepared by Bruce Babson, Research Fellow at MicroConstants, Inc. in San Diego, California, for the CACO-PBS Mini-Symposium on Bioanalytical and Analytical Applications and Problem Investigation Case Studies. Bruce is one of twelve presenters at the August 10, 2012 event in Foster City, California.

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A Novel Approach to Internal Standardization in LC/MS/MS Analysis; Sensitive LC/MS/MS Analysis of Gentamicin | MicroConstants

  1. 1. © 2012 MicroConstants, Inc. All Rights Reserved. CACO-PBSS Mini-Symposium: Bioanalytical and Analytical Applications and Problem Investigation Case Studies August 10, 2012 A Novel Approach to Internal Standardization in LC/MS/MS Analysis SENSITIVE LC/MS/MS ANALYSIS OF GENTAMICIN
  2. 2. © 2012 MicroConstants, Inc. All Rights Reserved. Background & Introduction Gentamicin is an aminoglycoside antimicrobial agent produced by fermentation of Micromonospora purpurea or M. echinospora. It has a wide spectrum of antimicrobial activity. Gentamicin is not a single molecule but a complex of three major and several minor components. Gentamicins C1, C1a and C2 are the three major components of the drug complex. The C2 component consists of two stereoisomers (C2 and C2a). Structures of Gentamicin Components Molecular Weights and Approximate Composition Gentamicin C1a MW 449.5 10-35% Gentamicin C2 & C2a MW 463.6 25-55% Gentamicin C1 MW 477.6 25-50%
  3. 3. © 2012 MicroConstants, Inc. All Rights Reserved. The Analytical Challenge • Gentamicin is associated with severe side effects • Use is subject to therapeutic drug monitoring, especially in patients with renal impairment • Product is being developed for subcutaneous injection at surgical incision sites to prevent post surgery infections – High systemic exposure not intended and hopefully avoided – Circulating plasma levels likely to be very low relative to IM, IV or oral routes of dosing • LLOQ of 1.00 ng/mL requested • Very low sample volumes are available • Gentamicin is co-administered with Vancomycin requiring sample volume for a separate assay
  4. 4. © 2012 MicroConstants, Inc. All Rights Reserved. Approaches in the Literature • USP monograph. Pre-column labeling with o-phthalaldehyde (OPA). Methodology makes a somewhat flawed assumption for equal response of components as they have different number of reactive primary amines. • Similar OPA derivative with micellar electrokinetic chromatography and UV @ 340 nm • HPLC 2012 poster by Thermo with ion-paired RP-HPLC (HFBA, TFA) charged aerosol detection • Reversed phase ion-pairing (octane sulfonic acid) pulsed electrochemical detection • Reversed phase ion-pairing TFA and methanol and electrospray LC/MS/MS Pharmaceutical Product Analyses: • SPE with on-column derivatization with 1-fluoro-2,4-dinitrobenzene RP-HPLC ultraviolet absorbance at 365 nm • Derivatization with phenyl isocyanate and RP-HPLC UV • Fluorenylmethyloxycarbonyl chloride (FMOC) derivatization, UV 265 nm or fluorescence • Tissues: HPLC with post column derivatization with OPA and fluorescence detection limits 400 ng/gm • Hydrophilic interaction chromatography (HILIC) with MS/MS detection Bioanalytical Analyses of Plasma or Tissues:
  5. 5. © 2012 MicroConstants, Inc. All Rights Reserved. Our Approach • Common themes: Ion Pairing or Derivatization • No precedent in literature to achieve required 1.00 ng/mL LLOQ using small plasma volumes • Initial approach = LC/MS/MS • No isotopically labeled I.S. would ever be available – Even if a single isoform could be synthesized would it track the other isoforms? – Spent considerable time and effort with an analogue I.S Tobramycin (MW 467.51) • Ion – Paired SPE with HFBA pretreatment on micro-elution C18 followed by LC/MS/MS • Decided to quantify C2 and C2a together (same MW and not readily resolved chromatographically) Tobramycin
  6. 6. © 2012 MicroConstants, Inc. All Rights Reserved. Initial Results • Made considerable progress • Sensitivity looked promising • HFBA modifier in a HILIC-type separation • Attempted to include Vancomycin Vancomycin Gentamicin C1 Tobramycin (I.S.) Gentamicin C2 and C2a Gentamicin C1a 725.1 > 1306.1 478.3 > 322.15 468.3 > 324.15 464.3 > 322.15 450.3 > 322.15
  7. 7. © 2012 MicroConstants, Inc. All Rights Reserved. Results Over Time • Vancomycin was successfully validated using an analogue I.S. (Ristomycin). LLOQ was 0.500 ng/mL using 20 µL of plasma. • Gentamicin exhibited poor ionization stability in the source • Good progress, but intuition suggested the technique would not validate per FDA Guidance • Other options were pursued Vancomycin Gentamicin C1 Tobramycin (I.S.) Gentamicin C2 and C2a Gentamicin C1a 725.1 > 1306.1 478.1 > 321.9 468.1 > 323.9 464.1 > 321.9 450.1 > 321.9
  8. 8. © 2012 MicroConstants, Inc. All Rights Reserved. How About Derivatization with LC/MS/MS? 1. Needs to tag all 5 amines efficiently and robustly 2. Need a stable derivative, one not affected by moisture during formation or stability post reaction 3. Pre-column derivatization for simplicity... let’s not add unnecessary mechanical complexity 4. Derivative should impart added hydrophobicity allowing more selective clean-up and chromatographic options 5. Bigger, but not too big! With 5 amines to tag, things could get out of hand quickly. 6. Improves LC/MS/MS sensitivity and ionization consistency – potentially new product ions and greater yield could vastly improve overall response 7. I.S. tracking – could the correct derivative make an analogue I.S. track better? 8. Manageable excess – a derivatization reagent for which excess reagent would either not be a problem or be readily eliminated For this application, what are the attributes of a good derivative?
  9. 9. © 2012 MicroConstants, Inc. All Rights Reserved. We Gave it a Try… • Given the attributes of a good derivative (previous slide), could all the criteria be met using alkyl chloroformates? • Reaction on primary and secondary amines: • Explored both ethyl chloroformate and propyl chloroformate – Reaction is base-catalyzed and works in aqueous phase – Acetone is a good co-solvent • Have previous experience with the derivatization of Amikacin (related amino glycoside) using ethyl chloroformate • Only obvious initial disadvantage was ionization - chloroformate derivatives of Gentamicin can find sodium where it doesn’t exist! R RI NH RII O O C CI R RI N C ORII O HCI+ + Amine Alkylchloroformate Carbamate
  10. 10. © 2012 MicroConstants, Inc. All Rights Reserved. Gentamicin Form: C1a C2 & C2a C1 Original Parent 450 464 478 Derivative (no adduct) 880 894 908 Sodium Adduct 902 916 930 Potassium Adduct 936 933 947 Ammonium Adduct 897 911 925 Initial Outcome Direct MS Infusion of PCF Reaction Product parents m/z 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 955 960 % 0 100 GENT001 1 (0.208) Scan ES+ 5.04e7932.64 894.59 880.63 878.55 861.64 881.45 882.58 892.58 918.62 916.60 895.60 908.68 902.45 896.67 909.69 919.56 930.69 920.63 921.32 946.60933.65 934.53 935.66 947.67 948.62 What masses can we expect for a 5 X PCF reaction? PCF derivatives of isomeric forms C2 and C2a were not chromatographically resolved and were treated as a single compound: Extracted standard representing 50 ng/mL total Gentamicin Gentamicin C1 Tobramycin (I.S.) Gentamicin C2 and C2a Gentamicin C1a 925.4 > 329.3 915.4 > 317.2 911.4 > 315.25 897.4 > 301.25 3.68 80288 1.71 590068 3.56 51178 3.32 53262
  11. 11. © 2012 MicroConstants, Inc. All Rights Reserved. Proof of Concept… Does it Work? • Tested extraction of 50 µL of plasma and calibration range from 1.00 to 4,000 ng/mL • Calculations by external standard showed promise! GOOD NEWS Great assay BAD NEWS No internal standard Gentamicin C1a Gentamicin C2 and C2a Gentamicin C1
  12. 12. © 2012 MicroConstants, Inc. All Rights Reserved. The Solution • Stable labeled I.S. is synthesized by pre- derivatization with d7-PCF • I.S. is added at the beginning of extraction but is not derivatized within the assay • Gentamicin isoforms in samples are derivatized within the assay by non-labeled PCF R RI NH Cd7O O C Cl R RI N C OCd7 O + Amine Propyl-d7 chloroformate Propyl-d7 carbamate
  13. 13. © 2012 MicroConstants, Inc. All Rights Reserved. Final Outcome Note: Each isoform has its own I.S in the same relative abundance as the drug substance because they originate from the same reference material. Internal Standard for Gentamicin C1a MRM 932.6 > 315.35 Gentamicin C1a LLOQ at 0.58 ng/mL MRM 897.4 > 301.2 Internal Standard for Gentamicin C2 and C2a MRM 946.6 > 329.4 Gentamicin C2 and C2a LLOQ at 0.88 ng/mL MRM 911.4 > 315.2 Internal Standard for Gentamicin C1 MRM 960.6 > 343.4 Gentamicin C1 LLOQ at 0.54 ng/mL MRM 925.4 > 329.2
  14. 14. © 2012 MicroConstants, Inc. All Rights Reserved. Validation Summary Gentamicin C1 Concentration (ng/mL) Gentamicin C1a Concentration (ng/mL) Gentamicin C2 + C2a Concentration (ng/mL) 1.62 54.0 1,080 1.74 58.4 1,180 2.64 88.0 1,760 Mean 1.60 54.1 1,100 Mean 1.76 58.4 1,180 Mean 2.65 88.9 1,780 %CV 5.41 2.89 4.02 %CV 6.89 2.93 3.78 %CV 5.25 4.11 3.09 %DEV -1.23 0.185 1.85 %DEV 1.15 0.690 1.72 %DEV 0.379 1.02 1.14 Interday Quality Control Sample Data and Statistics Example Calibration Curves Final validated range was 2.00 – 5,000 ng/mL total Gentamicin
  15. 15. © 2012 MicroConstants, Inc. All Rights Reserved. Summary & Conclusions • Novel application of deuterium labeled derivatization reagent to make an ideal I.S. • Multiple benefits to chloroformate derivatization • Critical aspects for success: – Parallel extraction for analyte and pre-derivatized I.S. – Robust derivatization – analyte has to “catch up” with I.S. – Analyte conversion – labeled derivative cannot exchange for unlabeled derivative • Further applications: – General approach for other aminoglycoside antibiotics – Fluoro-β-alanine (FBAL) – Doxorubicin and metabolite Doxorubicinol
  16. 16. © 2012 MicroConstants, Inc. All Rights Reserved. Acknowledgements Scientific support • Noel Henderson • David (Del) Lewiston Reagent synthesis • Isotec (Sigma-Aldrich) Management • Gilbert Lam, Ph.D. • David Beyerlein Presentation • Heather Rold

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