Dr Laurent Decosterd, PhD Innovation & Development Unit,

Laboratory of Clinical Pharmacology, Service of Biomedicine, CHUV Lausanne

Tandem mass spectrometry techniques for the TDM of antifungals :

new perspectives and challenges

1st International Workshop on Clinical Pharmacology of Antifungal Drugs and Fungal Diseases

Berlin, April 26 , 2013

• Rationale and usefulness of TDM

• Principle of Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS)

• Multiplex analysis of antifungal drugs

• Selected examples of applications • Monitoring of antifungal drugs combination • Identification of altered metabolism of voriconazole • Detection of previous treatments

• Conclusions

Therapeutic Drug Monitoring (TDM)

Therapeutig Drug Monitoring (TDM) : Measurement of plasma concentration under treatment Dosage individualization based on plasma levels, targeting

a pre-defined range Better chances of achieving therapeutic efficacy Reduced risks of meeting toxicity

Candidate Drugs : Drugs administrated on the long-term (chronic conditions) Drugs administrated to critically ill patients, with limited

remaining therapeutic options (ICU). Significant inter-patient pharmacokinetic variability Low intra-patient pharmacokinetic variability Consistent concentration - efficacy / toxicity relationships

Therapeutic Drug Monitoring (TDM) General principles

• Should be considered in case of lack of early, or easily accessible pharmacodynamic surrogates

• Unexpected clinical toxicities • Less than expected clinical response • Drug-drug or drug-food interactions • Changes in patho-physiological state, that may

impair gastrointestinal, hepatic or renal function • Pregnancy and pediatric patients • Monitoring short-term adherence

Therapeutic Drug Monitoring (TDM)

• Antifungal drugs meet the overall criteria for the implementation of a routine TDM program

• Measurements …and interpretations !!! • Reliable analytical methods must be

available

Therapeutic Drug Monitoring (TDM)

Development of UPLC-tandem MS method for the assay of antifungals drugs

• → Results must be available for TDM interpretations within 24-48h hours

• → The analytical method must be:

• Rapid (and user-friendly) • Precise (to limit the number of replicates) • Accurate (obviously…)

Instrument of High or Ultra Performance Liquid Chromatography (HPLC, UPLC )

Solvents (MeCN, MeOH, H2O)

Auto-sampler (samples) Chromatography column

(drugs and metabolites separation)

Detector: UV, fluorescence electrochemical

MS, MS/MS

Data processing (PC)

High pressure pump (400- 1000 bar)

0.2- 0.4 ml/min

From HPLC column To the first quadrupole of the MS instrument

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)

The Ionisation Step

We observe (and actually measure) The transition m/z 307.1 [M+H+] → m/z 238

Selection of parent ion: (i.e. molecular mass

: m/z 307.1 (fluconazole)

N

NN N

OH

N

N

F

F 307.1

Selection of product ion (for instance: caracteristic fragment:

m/z 238 daughter mass for fluconazole)

N

NN

OH

F

F

+

238

(Triple Stage Quadripole Mass Spectrometer)

TSQ Quantum, Thermo Scientific Corporation

Ionisation

Strength and Limitations of Tandem Mass Spectrometry

Strength: ultimate selectivity (thanks to triple quadripole mass detection)

Limitations: Ionisation of the analyte may be influenced by the presence of endogenous matrix components co-eluting with the target analyte that may influence its ionisation: = the so-called MATRIX EFFECT (Achilles' heel) → This influence must be eliminated or at least normalised.

From HPLC column To the first quadrupole of the MS instrument

Ionisation step

Stable isotope-labeled internal Standards (I.S.) and IS calibration method

• The use of stable isotope-labeled internal standards is the first choice approach to minimize the influence of matrix effect due to endogenous components co-eluting with the analytes that affects signal intensity, • and hence, the accuracy and precision of a quantitative method. • Of particular importance when using electrospray ionisation mass spectrometry. • The potential influence of can be normalised using the corresponding stable isotope-labeled Internal Standards (deuterium, 13C).

From HPLC column To the first quadrupole of the

MS instrument

« Multiplex » Analyses of antifungal

drugs • Fluconazole • Voriconazole • Voriconazole-N-oxide • Posaconazole • Itraconazole • Hydroxy-itraconazole • Caspofungine • Anidulafungine

Antimicrob Agents Chemother, 54, 5302-5315 (2010)

Posaconazole

Hydroxy-itraconazole

Advantages : • Unique sample extraction procedure and short analytical

run. • Saves times by the establishment of simultaneous

calibration curves • Applicable for blood samples from patients receiving either

different single-drug, or combination regimens • Results obtained on a daily basis: TDM dosing adjustments

performed within shortest time intervals. • Detection of other drugs possibly present in sample.

« Multiplex » assay: Analysis of drugs from the same therapeutic

class in a single chromatographic run

Fluconazole

Fluconazole-d4

Voriconazole-NO

Voriconazole-NO-d3

Voriconazole

Voriconazole-d3

Caspofungin

Posaconazole

HO-itraconazole

HO-itraconazole-d5

Anidulafungin

Itraconazole

Itraconazole-d5 Caspofungin analogue

Posaconazole-d4

An Ultra-Performance Liquid Chromatography - tandem MS method for the simultaneous determination of 6 antifungal agents and their metabolites

The chromatographic separation of 8 compounds takes place in less than 5 minutes

2.7 µg/mL

Voriconazole N-oxide : in source metabolite dissociation → voriconazole

0.4 µg/mL

Chromatogram from an Acute Lymphoblastic Leukemia ( ALL) patient on oral voriconazole 400 mg BID. Voriconazole trough level was 0.4 µg/mL (below the proposed therapeutic range) Voriconazole N-oxide level was 2.7 µg/mL suggesting an accelerated hepatic metabolism with first pass effect on oral bioavailability

Importance of the chromatographic separation to avoid cross-talk !

2.6 µg/mL

1.9 µg/mL

2. Detection of previous treatment

MULTIPLEX ASSAY → 1. Monitoring of antifungals drugs

combination

1.0 µg/mL

3.0 µg/mL

0.5 µg/mL

Fluconazole stopped 50 days before sampling

0.16 µg/mL

Antimicrob Agents Chemother, 54, 5302-5315 (2010)

The simultaneous assay of voriconazole and voriconazole-NO is an index of the phenotyping

activity of CYP2C19, that integrates : genetic background, liver function,

and drugs interactions

Hyland, R., B. C. Jones, and D. A. Smith. 2003. Identification of the cytochromeP450 enzymes involved in the N-oxidation of voriconazole. Drug Metab. Dispos. 31:540–547.

MULTIPLEX ASSAY → 3. High voriconazole levels

with impaired liver metabolism C:\Xcalibur\...\Fong-b205\fong-b205_018 2/27/2013 5:13:00 PM P 6676

RT: 0.00 - 5.10 SM: 15B

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Time (min)

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RT: 2.48AA: 1101028

RT: 2.93AA: 6744097

RT: 2.93AA: 12262203

NL: 3.12E5TIC F: + c ESI SRM ms2 366.100 [223.850-224.350] MS ICIS fong-b205_018

NL: 1.54E6TIC F: + c ESI SRM ms2 351.100 [280.950-281.450] MS ICIS fong-b205_018

NL: 2.90E6TIC F: + c ESI SRM ms2 353.100 [283.950-284.450] MS ICIS fong-b205_018

Voriconazole-NO

Voriconazole

Voriconazole-d3

7.2 µg/mL

4. Increased voriconazole metabolism as evidenced by High voriconazole-NO levels

C:\Xcalibur\...\Fong-b220\fong-b220c_020 4/15/2013 8:36:52 PM P 6894

RT: 0.00 - 5.10 SM: 15B

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RT: 2.20AA: 9319906

RT: 2.66AA: 1192749

RT: 2.65AA: 10449538

NL: 2.56E6TIC F: + c ESI SRM ms2 366.100 [223.850-224.350] MS ICIS fong-b220c_020

NL: 2.97E5TIC F: + c ESI SRM ms2 351.100 [280.950-281.450] MS ICIS fong-b220c_020

NL: 2.57E6TIC F: + c ESI SRM ms2 353.100 [283.950-284.450] MS ICIS fong-b220c_020

Voriconazole-NO

Voriconazole

Voriconazole-d3

2.0 µg/mL

0.8 µg/mL 7.8 µg/mL

How accurate is the multiplex assay ? → External Quality Control Program

KKGT, Stichting Kwaliteitsbewaking Klinische Geneesmiddelanalyse en Toxicologie: Association for Quality Assessment in TDM and clinical Toxicology, The Hague, The Netherlands. • International Interlaboratory External Quality Assurance Program for antifungals agents (organized at present for azoles and flucytosine)

International Interlaboratory External Quality Assurance Program for antifungals agents (57 labos):

LAUSANNE CHUV Results for 2012

Pascual A, Csajka C et al & Marchetti O, Clin Infect Dis 2012

Probability of response to voriconazole (solid line) and of grade 3 neurotoxicity (dashed line)

as a function of voriconazole trough plasma concentrations in adult patients with invasive fungal infections

→Therapeutic range : 1.5 to 4.5 mg/L

• Restricted to analytes contained in the calibration samples. • Total number of drugs in calibration is not infinite, because of solubility

issues of drugs in the matrix-match calibration samples, especially for lipophilic drugs at high concentrations (requiring the addition of non-matrix components (i.e. solvents) : < 10% of total biological matrix volume of calibrators.

• Calibration curves established for each analytical series, but used only in case of patients’ samples.

• Possibly requires an additional step of validation. • i.e. cross-validation calibration: « single analyte » versus

« multiplex » analysis. • Important in case of co-elution of analytes to exclude the occurence

of reciprocal competition at the ionisation step. • Easily circumvented, if stable isotopically labelled (D,13C,) Internal

Standards of drugs are available.

« Multiplex » assay: LIMITATIONS

LC tandem MS assay of further antifungal drugs

• Isavuconazole • Micafungin • Amphotericin B (free and total levels and

liposomal fraction) • New antifungal agents of current and future

classes

• Availability to isotopically labelled (D, 13C) Internal Standards (IS) for echinocandins (and new antifungal agents is the future) is a recurring issues. Efforts for a facilitated access must be prioritized.

• The assay of amphotericin B (administered as liposomal formulation) is an additional analytical challenge.

• Total amphotericin B plasma levels (free + liposomal) (ie. after liposomal disruption with suitable methodology (i.e. detergent, sonication))

• Free plasma levels of amphotericin B

Tandem mass spectrometry techniques for the TDM of antifungals: challenges

• The concentration of antifungal drug measured in plasma is at present the best marker of exposure in patient, integrating both genetic and non-genetic (drug interactions, patients patho-physiology, etc) influences.

• Strong arguments exist for implementing TDM for antifungal drugs, based on patients plasma concentrations.

• Randomised trials of prospective TDM intervention integrating clinical outcome, emergence of resistance and morbidity/mortality are still lacking.

• In the emerging field of Personalized Medicine, the development of TDM for patient-tailored dosage adjustment of antifungal therapy should allow to maximize both the therapeutic benefit and the tolerability of drugs.

TDM of antifungals: Conclusions

“ The right dose of the right drug to the right patient.”

• Thomas Mercier • Dr Boris Zanolari • Beatrice Ternon • Sandra Cruchon • Nicole Guignard

• Manel Aouri, PhD student • Jaurès Blanc, PhD student • Dr Aurélie Fayet–Mello • Dr Nicolas Widmer

Acknowlegments

• Prof Oscar Marchetti • Prof Chantal Csajka • Prof Thierry Buclin • PD Dr Matthias Cavassini • Prof Amalio Telenti • Prof Chin Bin Eap

Laboratory Research Scientists

Partners within the Lausanne University Hospital

Financial Supports and Collaborations

Centre Coordonné d’Oncologie ambulatoire,

CCO Lausanne

Swiss Tropical Institute Basel