Importance of End-to-End Robustness when dealing with ... · as Impurity of O-glucuronide Naloxone O-glucuronide ... • 60 mg b.i.d Dextromethorphan Total Dextrorphan Cmax (ng/mL)

© 2014 inVentiv Health. All rights reserved.

Importance of End-to-End Robustness when dealing with Glucuronide Metabolites

Luc Bouchard M.Sc.

2 © 2014 inVentiv Health. All rights reserved.

Agenda

•  Introduction

•  Biological samples

•  Samples analysis

•  Samples extraction

•  Quantification of glucuronides ›  Indirect ›  Direct

•  Conclusions


Introduction

•  Glucuronidation is a major metabolic detoxification pathway ›  Formation of inactive water-soluble molecules Excretion ›  Metabolite more active than the parent drug Bioactivation

•  Conjugation process = Nucleophilic xenobiotic molecule + glucuronic acid

Functional Group (XH) Product Carboxylic acid R-COOH Ester Alcohol R-OH Ether Thiol R-SH Thioether Amine RNH2, R3N Aminoketal or 4° amine

Hydrolysis Stability

-

+

β-D-glucuronide


Introduction

•  Bioanalytical assay robustness ›  Ability to behave under multiple conditions to generate reliable results.

Bioanalytical assay

Biological sample

Sample extraction

Sample analysis

(LC-MS/MS)

Results

•  Back-conversion of metabolites at every step of the assay must be evaluated ›  Improve reliability of the assay to deliver accurate concentration of

unconjugated analyte in incurred samples.


Biological sample

•  Evaluate the impact of the glucuronide metabolite on the measurement of unconjugated drug ›  Various stability tests in matrix in presence of metabolite.

Ezetimibe

80-90% of total drug as O-glucuronide (phenol group)

Tests Short-term stability in matrix Long-term stability in matrix

Freeze and thaw stability Post-preparative stability

Whole blood stability Incurred sample reanalysis

FN

OH

O

OH

F

•  Low and high QCs fortified with metabolite ›  Estimated Cmax concentration. ›  Estimated physiological concentration.


Biological sample: case study

•  Perindoprilat: incurred sample reanalysis inaccuracy

0

5

10

15

20

25

30

2 4 8

Varia

tion

(%)

Incubation time (h)

High concentration of metabolites

Low concentration of metabolites

•  Perindoprilat results were inaccurate for sampling times 0.3h to 3h ›  Back-conversion of metabolites into Perindoprilat in plasma samples. ›  Method was modified to avoid the back-conversion.

Incurred sample incubation (20°C) Low QC incubation (24h, 20°C)

N

H

H

O

CH3

NH

COOH

COOH

CH3

0

10

20

30

40

50

Varia

tion

(%)

Not fortified

Fortified with Perindopril acyl glucuronide

Fortified with Perindoprilat acyl glucuronide


Sample analysis (LC-MS/MS)

•  Evaluate the potential fragmentation of the metabolite at the detection step to avoid over-estimation of the parent concentration ›  MS ion source (ESI or APCI). ›  Collision cell (ion channel cross-talk).

•  Develop good chromatographic separation conditions of parent and metabolites ›  Avoids a false estimation caused by the fragmentation of the co-eluting

glucuronide metabolite. ›  Assess the chemical purity of the β-D-glucuronide reference standard in term

of parent analyte.


328 → 212 ESI +

Sample analysis (LC-MS/MS)

Naloxone + O-glucuronide (solution)

Naloxone

328 → 212 ESI +

In source fragmentation

Naloxone O-glucuronide (solution)

Naloxone as Impurity of O-glucuronide

Naloxone O-glucuronide

504 → 486 ESI +

O

N

O

CH2

OH

OH

Evaluation of fragmentation Assessment of purity


181 → 94 ESI +

Sample extraction

•  Evaluate the potential hydrolysis of β-D-glucuronide during samples preparation in the measurement of the unconjugated drug

PAcBA (human plasma)

PAcBA acyl glucuronide

(solution)

PAcBA

181 → 94 ESI +

NH

OH

O CH3O

In source fragmentation

of PAcBA Acyl glucuronide

No parent detected

Evaluation of fragmentation and purity


Sample extraction

Blank human plasma

•  Extraction conditions were modified to reduce the hydrolysis of the PAcBA glucuronide ›  Ensures accurate quantification of the parent analyte in incurred samples.

NH

OH

O CH3O

181 → 94 ESI +

No PAcBA detected

PAcBA acyl glucuronide (blank human plasma)

181 → 94 ESI +

PAcBA from hydrolysis of acyl glucuronide during extraction

process


Quantification of glucuronide metabolites

•  Determination of the concentration of glucuronides might be required ›  Indirect quantification

•  Hydrolysis of β-D-glucuronide prior to sample extraction to determine total concentration of drug (unconjugated + conjugated).

•  The glucuronide concentration is obtained by calculations (total – unconjugated).

•  Hydrolysis step could be fitted in the assay of the unconjugated analyte.

›  Direct quantification •  Extraction and analysis of intact glucuronide. •  Glucuronide metabolite must be stable. •  Total concentration of drug is obtained by summation of the unconjugated

drug and glucuronide. •  New assay needs to be developed.


Indirect quantification of glucuronide

•  Hydrolysis of glucuronides for indirect determination ›  Enzymatic (β-glucuronidase). ›  Chemical (acid or base).

•  Efficient hydrolysis conditions required to ensure accurate measurement of total concentration of drug ›  Monitoring of the hydrolysis yield is required.

•  Glucuronide hydrolysis may be incomplete ›  Binding to matrix components. ›  Reactivity varies between substrates and structural isomers.

•  Concentration of enzyme or reagents. •  Incubation temperature. •  Reaction time.

›  Parent analyte may be unstable in hydrolytic conditions.


Indirect quantification of glucuronide

•  Monitoring of the efficiency of hydrolysis within an analytical batch

Dextrorphan O-glucuronide

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

100.0

27 66 126 181 264 504 724 943 1381 1800 2583

Con

vers

ion

(%)

β-Glucuronidase Concentration (U/mL)

β-Glucuronidase from E.coli Human plasma Glucuronide conc. = 20 ng/mL Incubation: 1h, 50°C, pH = 7

Hydrolysis of Dextrorphan O-glucuronide

Calibration Quality control Approach 1 Unconjugated Glucuronide Approach 2 Glucuronide Glucuronide

O

NCH3

H

Glu

•  Conditions of the hydrolysis


Direct quantification of glucuronide

•  Dextrorphan O-glucuronide is stable ›  During and after the extraction process. ›  In plasma at room temperature (22h). ›  After freeze and thaw cycles (4 cycles). ›  In whole blood at 4°C (4h).

•  Direct analysis of intact Dextrorphan O-glucuronide by protein precipitation

Accuracy and Precision QC Conc.

(ng/mL) Accuracy Bias (%)

Precision CV (%)

Inter-assay (4 runs, N=24)

LLOQ 8 -3.61 4.94 Low 24 -1.32 4.77

Middle 4000 1.15 3.95 High 6000 -0.51 3.57

•  Incurred sample reanalysis (ISR) performance near 100%

O

NCH3

H

Glu


Determination of total Dextrorphan in human plasma

•  60 mg b.i.d Dextromethorphan Total Dextrorphan

Cmax (ng/mL)

Dextromethorphan Dextrorphan Total Dextrorphan Published NDA 3.7 15.9 733.4 inVentiv Health Clinical 5.6 11.4 1856

•  Published NDA results ›  Quantification of hydrolyzed Dextrorphan (no detailed protocol).

•  inVentiv Health Clinical ›  Quantification of intact Dextrorphan glucuronide.

•  Was the hydrolysis efficiency properly monitored for the results published in the NDA?


Conclusions

•  Glucuronide metabolites can hydrolyze back to parent drug

•  The fate of the glucuronide metabolite must be evaluated at each step of the assay ›  Sample

•  Stability in matrix.

›  Extraction •  Stability during and after extraction process.

›  Analysis •  Glucuronide fragmentation. •  Separation of metabolite and parent by chromatography.

•  Hydrolysis conditions must be monitored for indirect quantification of glucuronide

•  Glucuronide must be stable enough to be extracted and analyzed directly


Acknowledgements

•  Dr. Ann Levesque

•  Mr. Sylvain Lachance

•  Ms. Nadine Boudreau

•  R&D team (Quebec City)

•  Mr. Daniel Marquis

•  Mr. François Viel

Documents

Importance of End-to-End Robustness when dealing with ... · as Impurity of O-glucuronide Naloxone O-glucuronide ... • 60 mg b.i.d Dextromethorphan Total Dextrorphan Cmax (ng/mL)