Inter-laboratory Analysis of Isavuconazole Plasma Concentration Assays Among

European Laboratories

Federico Pea, MD,* Robert Krause, MD,† Carsten Müller, MD,‡ Benjamin Hennart, PharmD,§

Malcolm Richardson, PhD,¶ Andreas Meinitzer, PhD,† Martin H.J. Wiesen, MD,‡ Tatiana

Wiktorowicz, PhD,ǁ Jochen Spickermann, PhD,ǁ and Anne Santerre Henriksen, PhD,ǁ**

From the *Department of Medicine, University of Udine, and Institute of Clinical

Pharmacology, Santa Maria della Misericordia University Hospital, ASUIUD, Udine, Italy;

† Section of Infectious Diseases and Tropical Medicine, Department of Internal Medicine,

Medical University of Graz, Graz, Austria;

‡Center of Pharmacology, Department of Therapeutic Drug Monitoring, University

Hospital of Cologne, Cologne, Germany;

§Toxicology and Genopathy Unit, CHU Lille, Lille, France;

¶Mycology Reference Centre Manchester, ECMM Excellence Centre of Medical

Mycology, Manchester University NHS Foundation Trust, Manchester, UK;

ǁBasilea Pharmaceutica International Ltd., Basel, Switzerland

**Affiliation at the time of the study

Correspondence: Federico Pea, MD

Address: Piazzale Santa Maria della Misericordia, 3 – 3300 Udine, Italy

Telephone: +39 0432 559833

Fax: +39 0432 559819

(email: federico.pea@uniud.it)

(Address for reprints, as above)

Supported by Basilea Pharmaceutica International Ltd.

1

Disclosures

F.P. has received personal fees from Basilea Pharmaceutica, Gilead, MSD, and Pfizer

unrelated to the current study. R.K. has received research grants from Merck and served on

the speakers’ bureau of Pfizer, Gilead, Astellas, Basilea, and Merck. C.M. has received

personal fees from Astellas unrelated to the current study. M.R. has received grants and

personal fees from Basilea related to the current study and personal fees from Gilead

Sciences, Astellas, MSD, and Pfizer unrelated to the current study. T.W. and J.S. are full-

time employees of Basilea Pharmaceutica International Ltd. A. S-H. was a full-time employee

of Basilea Pharmaceutica International Ltd at the time of the study. B.H., A.M., and M.H.J.W.

have nothing to disclose.

2

Abstract

Background: Under certain circumstances, clinicians treating patients with isavuconazole

for invasive aspergillosis or mucormycosis may use therapeutic drug monitoring. However,

the accuracy and reproducibility of the various assays used by different laboratories for the

quantification of isavuconazole plasma concentrations have yet to be determined.

Methods: Human plasma samples spiked with known concentrations of isavuconazole were

provided to European laboratories that took part in a “round-robin” test (an inter-laboratory

test performed independently at least two times; two rounds performed in the current study).

Assay methods included liquid chromatography-tandem mass spectrometry (LC-MS/MS), LC

with ultraviolet detection (LC-UV), LC with fluorescence detection (LC-FL), and bioassay.

The accuracy and reproducibility compared with the known concentrations for each sample

in each round were compared overall, between assays, and between laboratories.

Results: Twenty-seven laboratories participated in the study (LC-MS/MS, n=15; LC-UV; n=9;

LC-FL, n=1; bioassay, n=2). In Round 1, for nominal concentrations of 1000, 1700, 2500,

and 4000 ng/mL, the mean (SD) determined concentrations were 1007 (183), 1710 (323),

2528 (540), and 3898 (842) ng/mL, respectively. In Round 2, for nominal concentrations of

1200, 1800, 2400, and 4000 ng/mL, the mean (SD) determined concentrations were 1411

(303), 2111 (409), 2789 (511), and 4723 (798) ng/mL, respectively. Over both rounds,

determined concentrations were consistently within 15% of the nominal concentrations for 10

laboratories (LC-MS/MS, n=4; LC-UV, n=5; bioassay, n=1) and consistently exceeded the

upper 15% margin for 7 laboratories (LC-MS/MS and LC-UV, n=3 each; LC-FL, n=1).

Conclusions: Alignment of methodologies among laboratories may be warranted to improve

accuracy and reproducibility of therapeutic drug measurements.

Key Words: Bioassay; isavuconazole; LC-FL; LC-MS/MS; LC-UV

3

INTRODUCTION

Triazole antifungal agents are an important class of drugs for the prophylaxis and

treatment of invasive fungal infections (IFIs), which are associated with high morbidity and

mortality in immunocompromised patients.1 However, maintenance of safe and efficacious

plasma or serum concentrations of these drugs can sometimes necessitate therapeutic drug

monitoring (TDM). Factors inherent to a drug that dictate the need for TDM include a high

level of intra- and inter-individual variability of pharmacokinetics (PK), a defined therapeutic

range, and a narrow therapeutic window.2

Isavuconazole is the active moiety of the prodrug isavuconazonium sulfate, the newest

available triazole antifungal agent with demonstrated efficacy and safety for treatment of

adults with IA or mucormycosis.12-14 An early phase study demonstrated good predictability of

the dose-exposure relationship,15 and in a population-PK analysis using data from the phase

III SECURE trial in adult patients with IA and other invasive mold diseases, no relationship of

exposure with either efficacy or elevations/abnormalities in liver enzyme test results was

found.16 This suggests that, in most adults, the clinical dose of isavuconazole results in

exposures that are within the optimal therapeutic window, and so routine TDM of

isavuconazole may not be necessary. Nevertheless, the margins of the therapeutic window

remain to be defined, and it has yet to be determined if and when TDM might be required

during isavuconazole treatment.

Reliable interpretation of the results of TDM requires that the analytical assays are both

accurate and reproducible. Several laboratories have published or presented methods for the

analysis of isavuconazole concentrations using liquid chromatography with tandem mass

spectrometry detection (LC-MS/MS),17-21 ultraviolet detection (LC-UV),22-24 or fluorescence

detection (LC-FL),25 or using bioassay.26 However, it has not been defined how the results of

such analyses compare, both with respect to the different analytical methods used and to the

inter-laboratory differences using the same techniques. Therefore, we conducted a round-

robin exercise (inter-laboratory test performed independently at least two times) at European

4

laboratories to assess the accuracy and reproducibility of measurements of isavuconazole

concentration measurements.

5

MATERIALS AND METHODS

This study commenced September 2016 and completed July 2017.

Preparation of Samples

All samples were prepared by Basilea Pharmaceutica. A 5 mg/mL stock solution was

prepared by dissolving 7.102 mg of isavuconazole in 1.284 mL of dimethyl sulfoxide

(DMSO). The stock solution was then diluted to prepare spiking solutions of 100,000,

170,000, 250,000, 400,000 ng/mL (Round 1) or 120,000, 180,000, 240,000, 400,000 ng/mL

(Round 2) in a 1/1 solution of DMSO/acetonitrile + 0.05% trifluoroacetic acid

(DMSO/ACN/TFA). Samples for the round robin were prepared by addition of 5 μL of the

spiking solutions to 495 μL samples of human plasma containing K3-EDTA as anticoagulant.

Final concentrations of samples used were based on the clinical relevant range of plasma

concentrations for isavuconazole. Concentrations in Round 1 were as follows: Sample 1,

1000 ng/mL; Sample 2, 1700 ng/mL; Sample 3, 2500 ng/mL; and Sample 4, 4000 ng/mL.

Concentrations in Round 2 were as follows: Sample 1, 1200 ng/mL; Sample 2, 1800 ng/mL;

Sample 3, 2400 ng/mL; and Sample 4, 4000 ng/mL. For each round, all plasma samples of a

given concentration were mixed together and re-aliquoted prior to distribution, such that each

laboratory effectively received the same sample.

Laboratories and Testing

European laboratories with the facilities for and expertise in drug-concentration assays

using LC-MS/MS, LC-UV, LC-FL, or bioassays based on diffusion of the drug into inoculated

and incubated media, were identified and invited to participate in the study. All laboratories

included in the study were required to have developed and validated the assays used in this

study prior to inclusion. Participating laboratories, listed in Supplementary Table S1, were

provided with samples in the first and/or second round of the test. Details of some of the

analytical methods used have been published previously17-19,22,24,25; others were adapted from

methods established for detection of other triazole antifungal agents,27-29 or used/adapted a

commercially available assay.30 All samples for each round were also tested by the Basilea

6

Pharmaceutica bioanalytical laboratory using LC-MS/MS. Details of methods used by Basilea

Pharmaceutica and as provided by each laboratory are provided in the Supplementary

Methods. Results from Basilea Pharmaceutica were included in statistical analyses but were

not considered when comparing different methods or laboratories.

Statistics

To determine the accuracy and precision of measurements made in each round, the

mean, standard deviation, and bias (percent deviation from nominal concentration) were

calculated for all samples and for each assay method. The results were assessed with

respect to the European Medicines Agency (EMA)31 and the US Food and Drug

Administration (FDA)32 guideline requirements of accuracy (bias) and precision (relative

standard deviation) within a 15% limit.

7

RESULTS

In total, 34 laboratories in Germany, UK, Netherlands, Italy, France, Austria,

Switzerland, and Denmark were invited to participate in the round-robin test. Of these, 27

laboratories provided results for the determination of isavuconazole concentrations in human

plasma (Supplementary Table S1; Round 1, Laboratories 1–10; Round 2, Laboratories 11–

27). The methods used for the determination were LC-MS/MS (Round 1: Laboratories 1–5;

Round 2: Laboratories 11–20), LC-UV (Round 1: Laboratories 6–8; Round 2: Laboratories

21–26), bioassays (Round 1: Laboratories 9, 10; Round 2, Laboratory 10), and LC-FL

(Round 2: Laboratory 27).

In Round 1, the mean of determined values for the assessments from all laboratories

for each of the samples with nominal concentrations of 1000, 1700, 2500, and 4000 ng/mL

were within the 15% margins, although the standard deviations extended beyond those

margins (Fig. 1). The overall biases at each of these concentrations were 0.7%, 0.6%, 1.1%,

and –2.5%, respectively. Among all 40 samples assessed by the 10 laboratories, 29

determined concentrations were within 15% of the nominal concentrations (acceptance

criterion), 7 were above the upper 15% margin, and 4 were below the lower 15% margin.

The results were also assessed as a function of the analytic method. For LC-MS/MS

(Laboratories 1–5), the mean of determined values for each nominal concentration was again

within the 15% margins, although the standard deviations exceeded the upper margin at all

tested concentrations (Fig. 2). Determined values from 2 laboratories were consistently

within the 15% margins and those from 1 laboratory consistently exceeded the upper 15%

margin. The overall biases at each of the tested concentrations (1000‒4000 ng/mL) were

4.3%, 7.8%, 9.5%, and 6.9%, respectively. In total, 13/20 determined concentrations were

within the 15% margins. The remaining 7/20 concentrations exceeded the upper 15%

margin, including 1 sample for Laboratory 2, 2 samples for Laboratory 4, and all samples for

Laboratory 5. Determined values from the 3 laboratories that used LC-UV were consistently

within 15% of the nominal concentrations (Fig. 2). The overall biases at each of the tested

concentrations were 7.3%, 3.9%, 7.5%, and 0.2%, respectively. Of the 2 laboratories that

8

used bioassays, all values determined from Laboratory 9 were within 15% of the nominal

concentrations, whereas those from Laboratory 10 were all well below the lower 15% margin

for all samples (overall bias at each concentration, –20%, –26.2%, –33.6%, and –34.5%,

respectively) (Fig. 2).

In Round 2, the mean of determined values for the assessments from all laboratories

slightly exceeded the upper 15% margin for each of the samples with nominal concentrations

of 1200, 1800, 2400, and 4000 ng/mL (Fig. 3). The overall biases at each of these

concentrations were considerably larger than those observed in Round 1 (17.6%, 17.3%,

16.2%, and 18.1%, respectively). Among all 72 determined concentrations from the 18

laboratories, 31 were within the 15% margins, 36 exceeded the upper 15% margin, and 5

were below the lower 15% margin.

When assessed as a function of the analytic methods, the means of determined values

for those using LC-MS/MS (Laboratories 11–20) were slightly below the upper 15% margin

(Fig. 4). Determined values from 2 laboratories were consistently within the 15% margins and

those from 2 other laboratories consistently exceeded the upper 15% margin. The overall

bias at each nominal concentration was again higher than observed for LC-MS/MS in Round

1 (14.4%, 13.9%, 11.6%, and 12.1%, respectively). In total, 19/40 determined concentrations

were within the 15% margins, 18/40 exceeded the upper 15% margin, and 3/40 were below

the bottom 15% margin. Among laboratories that used LC-UV (Laboratories 21–26), the

means of determined values for all nominal concentrations exceeded the upper 15% margin

(Fig. 4). Two laboratories consistently provided determined values that were within the 15%

of the nominal concentrations and 3 laboratories provided values that all exceeded the upper

15% margin. The overall bias at each nominal concentration was 25.6%, 24.3%, 28.6%, and

30.8%, respectively. Among the 24 total samples, 10 of the determined concentrations were

within the 15% margins and 14 exceeded the upper 15% margin. For LC-FL (Laboratory 27),

determined values for all samples exceeded the upper 15% margin (Fig. 4). For bioassay,

Laboratory 10 was included again after having made technical adjustments. Although

determined values for the 1200 ng/mL and 2400 ng/mL nominal concentrations were each

9

slightly below the lower 15% margin, determined values were with the 15% margins for the

1800 ng/mL and 4000 ng/L nominal concentrations (Fig. 4).

10

DISCUSSION

In any instance for which isavuconazole TDM might be considered appropriate, its

usefulness will require accurate measurement with regard to the analytic method used. In

this round-robin exercise of European laboratories, 10 of the 27 laboratories included in both

rounds provided determined plasma concentrations that were consistently within ±15% of the

nominal concentrations. Of all total samples in both rounds, 54% (60/112) determined

concentrations were within ±15% of the nominal concentrations. These data suggest that

there is likely need for refinement and alignment of each of those analytical methods across

the different laboratories.

It may be a concern that determined concentrations were consistently within the 15%

margins stipulated in guidelines from the EMA31 and FDA32 for fewer than half of the

laboratories. Moreover, determined concentrations outside of those margins were far more

likely to be overestimated (43/112) than underestimated (9/112). Overestimation in clinical

practice might result in a decision to reduce the dose and thereby pose a risk for less

efficacious treatment. From a methodological perspective, this suggests that a common

systematic error across a subset of the laboratories is possible. Data regarding the assay

calibrations performed were not available for all laboratories. As isavuconazole is a relatively

new drug, it may be that some have not yet implemented the adjustments necessary to

obtain the desired accuracy. For example, it is unclear whether all laboratories using LC

controlled for possible matrix effects during elution.33 Controls for matrix effects were

reported by 4 of the laboratories in the current study, and among those laboratories,

determined concentrations were consistently within the 15% margin for 3 (LC-MS/MS:

Laboratories 1,17 3,19 and 1818) but consistently exceeded the upper 15% margin for 1 (LC-FL:

Laboratory 2725). Another potentially important source of systemic error is failure to account

for the content of isavuconazole in locally-prepared, concentrated stock solutions for

preparation of calibration/quality control samples. Therefore, it seems likely that there is a

need for standardization of methods, including extraction and dilution of samples, use of

appropriate internal standards, working/stock solutions, and use of solvents. The use of an

11

internal standard would help to enhance the accuracy of the different assays used in this

study to varying degrees. The use of a stable labeled internal standard is best practice for

LC-MS/MS. For a LC-UV approach, an internal standard would need to be

chromatographically separated from all UV peaks of interest. However, for a bioassay

method, an internal standard would not be necessary for concentration determination.

It is noteworthy that the largest overestimations were observed with LC-UV in Round 2

and appeared to be pronounced at higher nominal concentrations. This may involve a

systematic error in purification steps (i.e., steps also used for LC-MS/MS), but that alone may

not account for the greater magnitude of bias. One potential explanation is that uncontrolled

ionization effects, which might have affected LC-MS/MS measurements by some

laboratories, might have an even greater effect on UV absorption. That might also explain the

overestimations observed using LC-FL, although no firm conclusions could be drawn

regarding the overall accuracy and reproducibility of this analytic approach based on results

from 1 laboratory.

As only 2 laboratories in the round-robin test used bioassay, it was also not possible to

draw any firm conclusions from those results. In Round 1, the determined concentrations of

all samples from one of the laboratories were all within the 15% margin; however, the second

laboratory provided values that were underestimated for all samples. In Round 2, after

consultation with the first laboratory and alignment of methodology, the second laboratory

provided results that were all much closer to the nominal concentrations. Nevertheless, 2 of 4

values were still below the lower 15% margin and so it may be that this approach is prone to

inter-user variability.

Although the second laboratory that used bioassay was provided an opportunity to

review and adjust their methodology, other laboratories in this study were not, and so that

might be considered a limitation of this study. In fact, multiple assessments by each

laboratory for each concentration might possibly have resulted in greater consistency.

However, this study was designed to more closely follow normal practice in a clinical setting,

during which replicates are not usually performed. It is also important to note that, although

12

at least one laboratory in this study had possible effects of commonly co-administered

drugs,17 it is not known whether all of the other laboratories had done the same.

CONCLUSION

Taken together, the results of this study suggest that improving the accuracy and

reproducibility of each method is very likely to require standardization of the methodologies

across analytical laboratories. This process would require more open discussion regarding

protocols and might be facilitated by implementing international standards. Ultimately, it

would increase the confidence of clinicians in the event that isavuconazole TDM appears

necessary.

ACKNOWLEDGMENTS

Isavuconazole has been co-developed by Astellas Pharma Global Development Inc.

and Basilea Pharmaceutica International Ltd. The study was funded by Basilea

Pharmaceutica International Ltd, Basel, Switzerland. The authors thank all personnel at the

participating analytical laboratories (see Supplementary Materials). Medical writing support

was provided by Ed Parr, PhD, CMPP, of Envision Scientific Solutions and funded by Basilea

Pharmaceutica International Ltd.

13

REFERENCES

1. Lass-Florl C. Triazole antifungal agents in invasive fungal infections: a comparative

review. Drugs 2011;71:2405-2419.

2. Lewis R, Brüggemann R, Padoin C, et al. Triazole antifungal therapeutic drug

monitoring. Presented at the Sixth European Conference on Infections in Leukaemia;

Sophia Antipolis, France. 2015

3. Luong ML, Al-Dabbagh M, Groll AH, et al. Utility of voriconazole therapeutic drug

monitoring: a meta-analysis. J Antimicrob Chemother 2016;71:1786-1799.

4. Brüggemann RJ, Donnelly JP, Aarnoutse RE, et al. Therapeutic drug monitoring of

voriconazole. Ther Drug Monit 2008;30:403-411.

5. Dolton MJ, McLachlan AJ. Voriconazole pharmacokinetics and exposure-response

relationships: assessing the links between exposure, efficacy and toxicity. Int J

Antimicrob Agents 2014;44:183-193.

6. Walsh TJ, Raad I, Patterson TF, et al. Treatment of invasive aspergillosis with

posaconazole in patients who are refractory to or intolerant of conventional therapy:

an externally controlled trial. Clin Infect Dis 2007;44:2-12.

7. Ashbee HR, Barnes RA, Johnson EM, et al. Therapeutic drug monitoring (TDM) of

antifungal agents: guidelines from the British Society for Medical Mycology. J

Antimicrob Chemother 2014;69:1162-1176.

8. Chau MM, Kong DC, van Hal SJ, et al. Consensus guidelines for optimising antifungal

drug delivery and monitoring to avoid toxicity and improve outcomes in patients with

haematological malignancy, 2014. Intern Med J 2014;44:1364-1388.

9. Hamada Y, Tokimatsu I, Mikamo H, et al. Practice guidelines for therapeutic drug

monitoring of voriconazole: a consensus review of the Japanese Society of

Chemotherapy and the Japanese Society of Therapeutic Drug Monitoring. J Infect

Chemother 2013;19:381-392.

10. Mousset S, Buchheidt D, Heinz W, et al. Treatment of invasive fungal infections in

cancer patients-updated recommendations of the Infectious Diseases Working Party

14

(AGIHO) of the German Society of Hematology and Oncology (DGHO). Ann Hematol

2014;93:13-32.

11. Tissot F, Agrawal S, Pagano L, et al. ECIL-6 guidelines for the treatment of invasive

candidiasis, aspergillosis and mucormycosis in leukemia and hematopoietic stem cell

transplant patients. Haematologica 2017;102:433-444.

12. Natesan SK, Chandrasekar PH. Isavuconazole for the treatment of invasive

aspergillosis and mucormycosis: current evidence, safety, efficacy, and clinical

recommendations. Infect Drug Resist 2016;9:291-300.

13. Maertens JA, Raad, II, Marr KA, et al. Isavuconazole versus voriconazole for primary

treatment of invasive mould disease caused by Aspergillus and other filamentous

fungi (SECURE): a phase 3, randomised-controlled, non-inferiority trial. Lancet

2016;387:760-769.

14. Marty FM, Ostrosky-Zeichner L, Cornely OA, et al. Isavuconazole treatment for

mucormycosis: a single-arm open-label trial and case-control analysis. Lancet Infect

Dis 2016;16:828-837.

15. Schmitt-Hoffmann A, Roos B, Maares J, et al. Multiple-dose pharmacokinetics and

safety of the new antifungal triazole BAL4815 after intravenous infusion and oral

administration of its prodrug, BAL8557, in healthy volunteers. Antimicrob Agents

Chemother 2006;50:286-293.

16. Desai AV, Kovanda LL, Hope WW, et al. Exposure-response relationships for

isavuconazole in patients with invasive aspergillosis and other filamentous fungi.

Antimicrob Agents Chemother 2017;61.

17. Fatiguso G, Favata F, Zedda I, et al. A simple high performance liquid

chromatography-mass spectrometry method for therapeutic drug monitoring of

isavuconazole and four other antifungal drugs in human plasma samples. J Pharm

Biomed Anal 2017;145:718-724.

18. Müller C, Gehlen D, Blaich C, et al. Reliable and easy-to-use liquid chromatography-

tandem mass spectrometry method for simultaneous snalysis of fluconazole,

15

isavuconazole, itraconazole, hydroxy-itraconazole, posaconazole, and voriconazole

in human plasma and serum. Ther Drug Monit 2017;39:505-513.

19. Toussaint B, Lanternier F, Woloch C, et al. An ultra performance liquid

chromatography-tandem mass spectrometry method for the therapeutic drug

monitoring of isavuconazole and seven other antifungal compounds in plasma

samples. J Chromatogr B Analyt Technol Biomed Life Sci 2017;1046:26-33.

20. Kovanda LL, Petraitiene R, Petraitis V, et al. Pharmacodynamics of isavuconazole in

experimental invasive pulmonary aspergillosis: implications for clinical breakpoints. J

Antimicrob Chemother 2016;71:1885-1891.

21. Farowski F, Cornely OA, Vehreschild JJ, et al. Quantitation of azoles and

echinocandins in compartments of peripheral blood by liquid chromatography-tandem

mass spectrometry. Antimicrob Agents Chemother 2010;54:1815-1819.

22. Wissen CP, Burger DM, Verweij PE, et al. Simultaneous determination of the azoles

voriconazole, posaconazole, isavuconazole, itraconazole and its metabolite hydroxy-

itraconazole in human plasma by reversed phase ultra-performance liquid

chromatography with ultraviolet detection. J Chromatogr B Analyt Technol Biomed

Life Sci 2012;887-888:79-84.

23. Cozzi V, Baldelli S, Castoldi S, et al. Development and validation of a

chromatographic ultraviolet method for the quantification of isavuconazole in human

plasma samples. Ther Drug Monit 2018;40:512-514.

24. Nannetti G, Pagni S, Palu G, et al. A sensitive and validated HPLC-UV method for the

quantitative determination of the new antifungal drug isavuconazole in human

plasma. Biomed Chromatogr 2018:e4333.

25. Jørgensen R, Andersen SR, Astvad KMT, et al. Implementation of isavuconazole in a

fluorescence-based high-performance liquid chromatography kit allowing

simultaneous detection of all four currently licensed mold-active triazoles. mSphere

2017;2.

16

26. Rautemaa-Richardson R, Moore C, Richardson M. Isavuconazole levels in non-

immunocompromised patients treated for chronic pulmonary aspergillosis. Presented

at the 27th European Congress of Clinical Microbiology and Infectious Diseases;

Vienna, Austria. 2017

27. Gordien JB, Pigneux A, Vigouroux S, et al. Simultaneous determination of five

systemic azoles in plasma by high-performance liquid chromatography with ultraviolet

detection. J Pharm Biomed Anal 2009;50:932-938.

28. Jourdil JF, Tonini J, Stanke-Labesque F. Simultaneous quantitation of azole

antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional

high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B

Analyt Technol Biomed Life Sci 2013;919-920:1-9.

29. Franceschi L, D’Aronco S, Furlanut M. Development and validation of a liquid

chromatography - tandem mass spectrometry method for the determination of

voriconazole and posaconazole in serum samples from patients with invasive

mycoses. J Bioanal Biomed 2011;3:92-97.

30. Chromsystems Instruments and Chemicals GmbH. TDM of Isavuconazole using

MassTox® TDM Series A Kit. Available at: https://www.chromsystems.com/tdm-of-

isavuconazole. Accessed August 10, 2018.

31. European Medicines Agency. Guideline on bioanalytical method validation. Available

at: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/

2011/08/WC500109686.pdf. Accessed November 2, 2017.

32. US Food and Drug Administration. Guidance for Industry: Bioanalytical method

validation. Available at:

https://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf. Accessed

November 2, 2017.

33. Gosetti F, Mazzucco E, Zampieri D, et al. Signal suppression/enhancement in high-

performance liquid chromatography tandem mass spectrometry. J Chromatogr A

2010;1217:3929-3937.

17

18

Figure legends

FIGURE 1. Isavuconazole plasma concentrations determined by Basilea and all laboratories

in Round 1 (Labs 1–10) as a function of the nominal concentrations of 1000, 1700, 2500, and

4000 ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations,

dashed lines represent ±15% margins from nominal concentrations.

FIGURE 2. Isavuconazole plasma concentrations in Round 1 determined using liquid

chromatography-tandem mass spectrometry (LC-MS/MS; Labs 1–5), LC with ultraviolet

detection (LC-UV; Labs 6–8), or Bioassay (Labs 9, 10) as a function of the nominal

concentration of 1000, 1700, 2500, and 4000 ng/mL (A, B, C, and D, respectively). Solid

lines represent nominal concentrations, dashed lines represent ±15% margins from nominal

concentrations.

FIGURE 3. Isavuconazole plasma concentrations determined by Basilea and all laboratories

in Round 2 (Labs 10–27) as a function of the nominal concentrations of 1200, 1800, 2400,

and 4000 ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations,

dashed lines represent ±15% margins from nominal concentrations.

FIGURE 4. Isavuconazole plasma concentrations in Round 2 determined using liquid

chromatography-tandem mass spectrometry (LC-MS/MS; Labs 11–20), LC with ultraviolet

detection (LC-UV; Labs 21–26), LC with fluorescence detection (LC-FL; Lab 27), or Bioassay

(BA; Lab 10) as a function of the nominal concentration of 1200, 1800, 2400, and 4000

ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations, dashed

lines represent ±15% margins from nominal concentrations.

19

FIGURE 1. Isavuconazole plasma concentrations determined by Basilea and all laboratories

in Round 1 (Labs 1–10) as a function of the nominal concentrations of 1000, 1700, 2500, and

4000 ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations,

dashed lines represent ±15% margins from nominal concentrations.

20

FIGURE 2. Isavuconazole plasma concentrations in Round 1 determined using liquid

chromatography-tandem mass spectrometry (LC-MS/MS; Labs 1–5), LC with ultraviolet

detection (LC-UV; Labs 6–8), or Bioassay (Labs 9, 10) as a function of the nominal

concentration of 1000, 1700, 2500, and 4000 ng/mL (A, B, C, and D, respectively). Solid

lines represent nominal concentrations, dashed lines represent ±15% margins from nominal

concentrations.

21

FIGURE 3. Isavuconazole plasma concentrations determined by Basilea and all laboratories

in Round 2 (Labs 10–27) as a function of the nominal concentrations of 1200, 1800, 2400,

and 4000 ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations,

dashed lines represent ±15% margins from nominal concentrations.

22

FIGURE 4. Isavuconazole plasma concentrations in Round 2 determined using liquid

chromatography-tandem mass spectrometry (LC-MS/MS; Labs 11–20), LC with ultraviolet

detection (LC-UV; Labs 21–26), LC with fluorescence detection (LC-FL; Lab 27), or Bioassay

(BA; Lab 10) as a function of the nominal concentration of 1200, 1800, 2400, and 4000

ng/mL (A, B, C, and D, respectively). Solid lines represent nominal concentrations, dashed

lines represent ±15% margins from nominal concentrations.

23

Supplementary materials

TABLE S1. Laboratories Participating in the Round-Robin Test

Laboratory Country Method Round

1 Italy LC-MS/MS 1

2 Italy LC-MS/MS 1

3 France LC-MS/MS 1

4 France LC-MS/MS 1

5 France LC-MS/MS 1

6 Netherlands LC-MS/MS 1

7 Italy LC-UV 1

8 France LC-UV 1

9 United Kingdom Bioassay 1

10 Germany Bioassay 1 and 2

11 Italy LC-MS/MS 2

12 Italy LC-MS/MS 2

13 France LC-MS/MS 2

14 France LC-MS/MS 2

15 France LC-MS/MS 2

16 France LC-MS/MS 2

17 France LC-MS/MS 2

18 Germany LC-MS/MS 2

19 Germany LC-MS/MS 2

20 Austria LC-MS/MS 2

21 Italy LC-UV 2

22 Italy LC-UV 2

23 Italy LC-UV 2

24 France LC-UV 2

25 France LC-UV 2

26 United Kingdom LC-UV 2

27 Denmark LC-FL 2

LC-FL, liquid chromatography with fluorescence detection; LC-MS/MS, liquid

chromatography with tandem mass spectrometry; LC-UV, liquid chromatography with

ultraviolet detection

24

Supplementary Methods

Details of methods as provided by participating laboratories

Details of methods have been published previously for Laboratories 1,17 3,19 6,22 18,18 22,24

and 27.25

Liquid chromatography – tandem mass spectrometry (LC-MS/MS )

Basilea: Spiking solutions were prepared by serial dilution in DMSO and acetonitrile (ACN),

0.05% trifluoracetic acid (TFA) out of a 2 mg/mL stock solution with a range of 0.01–10

µg/mL. Calibration samples were performed by spiking 1 µL of each DMSO/ACN (0.05%

TFA) solution in 99 µL of blank K3-EDTA plasma. Each sample was then quenched with 300

µL of ACN, 0.05% TFA containing 1 µg/mL isavuconazole-d4 as internal standard. After

centrifugation, 10 µL of the supernatant was injected into the LC-MS/MS device. The same

preparation method was performed for the quality control samples. 50 µL of the study

samples were mixed with 150 µL of ACN, 0.05% TFA containing 1 µg/mL isavuconazole-d4

as internal standard and then treated like the calibrators and quality control samples. The

LOQ was 0.01 µg/mL and the linear range of the assay was 0.01–10 µg/mL. The coefficient

of variation ranged from 1.3% to 5.9% in the first round and 1.8% to 2.5% in the second

round of the round-robin test.

Laboratory 2: Working solutions of isavuconazole and IS were prepared by diluting each

analyte in DMSO. Calibrators and quality controls (QCs) were made by spiking analytes from

different batches of working solution into a pool of plasma. An 8-point calibration curve was

prepared by adding isavuconazole to yield concentrations that ranged from 0.08 to 10 mg/L

and included the lower limit of quantification (LLOQ). QC samples were prepared by adding

isavuconazole at the following 4 concentration levels (LLOQ, QClow, QCmedium and QChigh) to a

pool of plasma: 0.08, 0.31, 1.25, 5 ng/mL. A 50 µL aliquot of plasma was spiked with 10 µL of

10 µg/mL IS. Proteins were precipitated by adding 25 µL of extraction buffer and 250 µL of

precipitation reagent, vortexed and centrifuged for 10 minutes at 14,000 rpm. Supernatant

was then diluted 1:10 with methanol and transferred in an autosampler vial. LC-MS/MS

25

analysis was performed on a TSQ Quantum Access Max™ Triple Quadrupole coupled to an

Accela 1250 UHPLC system (ThermoFisher Scientific, Milan, Italy). Gradient separation

chromatography was carried out using a Accucore Polar premium analytical column 50 mm x

2,1 i.d. x 2.6 µm particle size (ThermoFisher Scientific, Milan, Italy) with mobile phase 1 and

2. The percentage of mobile phase 2 started at 20%, then programmed to reach 100% in 1.5

minutes and kept for 1 minute at flow rate of 600 µL/min, then the column was reconditioned

at 20% B for 2.5 minutes for a total run time of 5 minutes. The column temperature was

maintained at 40°C and injection volume was 2 µL. Ionization was achieved using HESI in

the positive ion mode with 3500 V. Nitrogen was used as the nebulizer and auxiliary gas, set

at 60 and 20 arbitrary units, respectively. Vaporizer and ion transfer tube temperature

settings were 350°C and 400°C. For collision-induced dissociation, high purity argon was

used at a pressure of 1.5 mTorr. Analytes were detected using selective reaction monitoring

(SRM) of the specific transitions for isavuconazole 438.2->127; 215; 224; 369 and its IS

442.2-> 127; 219; 224; 373 m/z.

Laboratory 4: Briefly, protein precipitation was achieved with the addition of 100 μL of

precipitation reagent [methanol/0.2 M zinc sulfate (80/20, v/v)] including 1 mg/L deuterated

isavuconazole (internal standard) in a 1.5 mL conical plastic Eppendorf test tube. Samples

were immediately vortexed and then centrifuged at 13,000 × g for 15 minutes at +4 °C. Then,

100 μL of supernatant was transferred into a sample vial and 10 μL was injected into the

chromatographic system. The chromatographic system consisted of Agilent (Palo Alto, CA,

USA) 1200 Series components including a binary pump, isocratic pump, column oven, 2 ten-

port switching valves and an autosampler. The hardware configuration included an Applied

Biosystems (Foster City, CA, USA) API 3200 equipped with a TurboIonSpray source. On-line

extraction was performed using a perfusion column (POROS R1/20, 2.1 mm × 30 mm,

Applied Biosystems, Foster City, CA, USA). The HPLC column was a short phenyl-hexyl

column (Phenomenex Luna 5 μm Phenyl-Hexyl, 2 mm × 50 mm, Aschaffenburg, Germany).

Data analysis was performed using the Analyst 1.4.2 software package (Applied Biosystems,

Foster City, CA, USA).

26

Laboratory 5: An LC-MS/MS method was developed and validated according to the

guidelines on bioanalytical method validation from the European Medicines Agency (2012)

for 6 azole analytes: fluconazole, voriconazole, posaconazole, isavuconazole, itraconazole,

and its active metabolite hydroxyl-itraconazole.

Laboratory 11: The LC-MS/MS method was adapted from a previously published approach

for detection of triazole antifungal agents.29

Laboratory 12: The LC-MS/MS method was based on a commercially-available approach

from Chromsystems.30 Sample preparation and subsequent analysis in LC-MS/MS were

performed using Chromsystems' parameter set Antimycotic drugs on Waters TQD

instrument. The Internal Standard Mix (800 μL) was added to 12 mL Precipitation Reagent to

form mixture A. A 50 μL sample/calibrator/MassCheck® control was pipetted into a 1.5 mL

reaction vial, to which 25 μL Extraction Buffer was added, mixed, and incubated for 2

minutes at ambient temperature (without centrifuging). 250 μL of mixture A was added and

mixed (vortex) for 30 seconds minimum before centrifuging 5 minutes at 15,000 x g. The

supernatant was diluted with Dilution Buffer prior to injection depending on the instrument

sensitivity. 0.2–50 μL was injected into the LC-MS/MS device (electrospray ionization;

multiple reaction monitoring mode; gradients, 0.00–0.50 min 30% mobile phase 2, 0.51–2.80

min 100% mobile phase 2, 2.81–3.20 min 30% mobile phase 2). The limit of quantification

ranged from 0.01 to 2.00 mg/L (linearity up to 250 mg/L; recovery of 95–111%; intra-assay

coefficient of variation [CV], 1.7–4.9%; inter-assay CV, 3.6–5.3%).

Laboratory 13: The LC-MS/MS method was based on a commercially-available approach

from Chromsystems.30

Laboratory 14: The LC-MS/MS method was adapted from a commercially-available

approach from Chromsystems.30 For the working solution of the internal standard, its

concentration was fixed at a concentration of 10 mg/L and 35 µL of the internal standard

working solution was added to a volume of 50 µL of sample. Sample preparation was

27

performed according to the protocol provided by Chromsystems and using extraction buffer

and precipitation reagent commercialized by Chromsystems.

Laboratory 15: The LC-MS/MS method was adapted from a previously published approach

to analysis of other triazole antifungal agents.28

Laboratory 16: LC-MS/MS was performed with deuterated internal standard extraction by

precipitation home-made standard curve.

Laboratory 17: The method was LC-MS/MS (HPLC- Quattro™ micro API detector, Waters,

MA, USA) with ESI positive mode. Samples were extracted with SPE MCX µElution

cartridge. The linearity was 0.2–8 µg/mL.

Laboratory 19: Extraction was from 200 µL serum/plasma, diluted with 200 µL 0.1 M

phosphate buffer pH 9.5, extracted with 1 mL chlorbutane/ether (1:1). Measurement was

performed with Agilent LC-TOF MS, internal standard Diazepam-d5. Calibration 2.5–25

mg/L.

Laboratory 20: Isavuconazole was measured with electrospray ionization tandem mass

spectrometry on a Voyager TSQ Quantum triple quadrupole instrument equipped with an

UltiMate 3000 chromatography system (Thermo Instruments, San Jose, CA, USA).

Chromeleon Xpress software for device management and LCQuanTM 2.7 for data

processing were used. Isavuconazole and its deuterated isotope were kindly provided by

Basilea (Basel, Switzerland). Calibrators and controls were prepared by spiking drug free

plasma from healthy donors with stock solutions. In brief, 20 µL serum samples (calibrators

or controls) were deproteinized by addition of ice cold 100 µL of methanol containing the

internal standard (isavuconazole-d4, 1000 ng/mL). After vortexing and centrifugation at

24,000 g (5 min), the clear supernatant was 10-fold diluted with 5% ACN. 10 µL were loaded

on a trapping column (POROS™ R1 20, 2.1 x 30 mm, Thermo Fisher) with mobile phase 1

(5:95 v/v ACN/water). After a short washing period the analytes were transferred and

separated on a Luna 5 μm Phenyl-Hexyl column 100A 50 x 2.1 mm (Phenomenex™

Aschaffenburg, Germany) with a linear gradient of mobile phase 2 (0.1% formic acid in MS

28

grade water) and mobile phase 3 (0.1% formic acid in MS grade ACN). Isavuconazole and

internal standard were monitored in positive multiple reaction monitoring mode using

characteristic precursor–product ion transitions: m/z 438.1→214.9 and m/z 442.1→218.9,

respectively. Within-day CVs were 2.9% (2900 ng/mL) and 1.5% (6600 ng/mL), and

between-day CVs were 3.1% (2900 ng/mL) and 1.8 % (6600 ng/mL). On the lower limit of

quantification (100 ng/mL) the coefficient of variation (CV) was <10%.

Liquid chromatography – ultraviolet detection (LC-UV)

Laboratory 7: Samples were prepared containing 0.25 mL plasma, 0.05 mL IS (6,7-

dimethylquinoxaline, 1 mg/L), 0.4 mL ACN, vortexed, then after 10 minutes they were

centrifuged at 17,000 rpm for 10 minutes and the supernatant transferred for drying under

nitrogen flow. Samples were reconstituted with mobile phase (0.2 mL; A: 50% monobasic

ammonium phosphate [NH4H2PO4] 0.04 M, pH=6; B: 50% ACN [CH3CN]) and 0.05 mL was

injected. UV detection was performed at 270 nm (linearity: 100–8000 ng/mL [calibration

curve with 7 not-zero points] plus 3 QC [320, 3200, 6000 ng/mL]).

Laboratory 8: This approach used LC with diode array detection. Plasma (200 µL), or

control, or calibrant, was mixed and centrifuged (10,000 g, 4°C, 10 minutes) after addition of

400 µL ACN for protein precipitation and containing 1.35 mg/L methyl-clonazepam (Sigma

Aldrich) as an internal standard. Supernatant (400 µL) was dried under nitrogen stream and

150 µL of a mix of ACN and phosphate buffer (pH 3.6) was added. Seventy microliters was

injected onto an UPLC-UV system (Acquity, Waters, Milford, MA) equipped with a

SYMMETRY 18 column (5 µM, 4.6 mm x 250 mm) at 30°C. Isavuconazole was detected at

284 nm and IS at 310 nm.

Laboratory 21: Briefly, 0.8 mL of ACN was added to 0.5 mL plasma samples. After protein

precipitation, the supernatant was dried; the residue was reconstituted in mobile phase and

injected into the HPLC device. Chromatographic separation was performed with a

SymmetryShield RP8 (3.9 mm × 150 mm, particle size 3.5 µm; Waters, Milan, Italy) column

29

under gradient conditions. The total chromatographic run time was 10 minutes per samples.

The method was linear in the range of 0.15–10 mg/L isavuconazole concentrations. The

lowest limit of quantification was set at 0.15 mg/L.

Laboratory 23: In a sensitive, selective high-performance liquid chromatographic (HPLC)

method with UV detection developed and validated for the determination of isavuconazole,

an aliquot (0.5 mL) of plasma or standard was pipetted into a 5 mL polypropylene tube

(Sarstedt, Leicester, UK) and ACN, 1.0 mL, added. The mixture was vortexed and the

mixture was centrifuged at 1200×g for 10 minutes. The organic layer was evaporated to

dryness and the residue was reconstituted with 500 μL of mobile phase and 180 μL injected

in the chromatographic system. The compounds were separated on a C18 5μm 150 × 4.6

mm (Restek) column using sodium potassium phosphate buffer (0.04 M): ACN ultrapure

(50/50 v/v) as mobile phase. The compounds were detected at a wavelength of 270 nm. The

assay was linear and validated over the range 0.25–8.0 mg/L. The mean recovery was 98%.

Assay reproducibility was: intraday <6%; interday <10%.

Laboratory 24: The LC-UV method was adapted from a previously published approach to

analysis of other triazole antifungal agents.27

Laboratory 25: The protocol used a liquid chromatographic method coupled with a photo

array diode for the detection. The calibration was made by standard from 0.25 to 10 mg/L.

We had 3 internal controls (1.5, 3.5 and 7.5 mg/L). The first step of the method consisted of

an extraction. 200 µL of sample added to 70 µL of Internal Standard (ketoconazole at 1

mg/L), 250 µL of ammonium acetate at 200 mM (pH9). After 10 seconds of mixing, 3.5 mL of

ethyl acetate/diethyl ether (50/50) was added, vortexed for 5 minutes, and the supernatant

was transferred and evaporated under nitrogen flux at 45°C. The dry extract was then

solubilized in 100 µL of a mixture of ACN/water (85/15). The chromatography system was a

UFLC-XR Shimadzu. A Kinetex C8 column (100 × 3 mm, 2.6 µm). 10 µL was injected in the

system. The chromatograms were extracted at 260 nm.

30

Laboratory 26: The LC was performed with a Gilson System (Autosampler, 231XL; Sample

Dilutor, 402; Pump, 307; Detector, 155 UV/VIS; System Interface 506C) with analytical

column (ThermoScientific, Accucore XL C18, 4 µm, 100 × 4.6 mm; mobile phase - ACN/0.2%

acetic acid 60/40). A 15 µL injection volume was used (ambient temperature for both the

sample and column) and detection was performed at 262 nm. Extraction was performed

using 250 µL serum + 50 µL internal standard (itraconazole) + 2 mL heptane/isoamyl alcohol

(98.5/1.5), rotated for 20 minutes, centrifuged for 5 minutes, supernatant removed, and then

concentrated under nitrogen for about 10 minutes until the tubes were dry before re-

dissolving in mobile phase.

Bioassay

Laboratory 9: Quality control samples with known amounts of isavuconazole were placed on

each bioassay plate, prepared as follows: A 1000 mg/L standard (A) was used as a drug

stock solution. This stock was diluted 1:10 (100 µL drug stock and 900 µL plasma) to give a

100 mg/L solution (B). For the 2 mg/L internal standard, stock solution B was further diluted

1:50 (150 µL drug stock and 7.35 mL plasma). For the 0.75 mg/L internal standard, stock

solution B was diluted 1:10 (100 µL drug stock and 900 µL plasma) to give a 10 mg/L stock

solution, which was then further diluted 1:13.3 (500 µL drug stock and 6.167 mL plasma).

Both sets of internal standards were then stored in 200 µL amounts in Z5 bottles at –80º C

for up to 3 months.

In the isavuconazole bioassay procedure, a 1000 mg/L stock solution of isavuconazole in

dimethyl sulfoxide (1000 mg/L) was prepared and dispensed into twelve 250 µL aliquots. A

hot plate was used to melt 90 mL of base agar before allowing it to cool to 56 ± 2°C in a

warm water bath. A solution of yeast nitrogen base with glucose and tri-sodium citrate

(YNBG + citrate) was also warmed to the same temperature in the bath. A suspension of

Candida kefyr (San Antonio strain) was prepared in 7 mL of sterile distilled water equal to 5

McFarland standard. The optical density of the suspension was determined at a wavelength

31

of 490 nm on a spectrophotometer in triplicate; the average optical density was calculated,

adjusted to an OD of 0.1, and the suspension was vortexed before removing 5 mL to a new

sterile universal to be used as the bioassay inoculum. When the agar had cooled to 56 ± 2°C

(approximately 30 minutes), the 10 mL aliquot of concentrated YNBG + citrate solution and

the 5 mL Candida kefyr suspension were vortexed thoroughly and added. The agar, YNBG +

citrate and Candida kefyr suspension was mixed before pouring it in to a labeled bioassay

plate ensuring no bubbles were present and left to solidify for at least 30 minutes. To

construct a standard curve, a set of standards of isavuconazole stock solution diluted in

plasma were prepared (0.125, 0.25, 0.5, 1.25, 2.5, 5, and 7.5 mg/L). When the agar had

solidified, cut 36 wells of 8 mm in diameter with sterile cork borer No. 3 were cut (6 rows of 6)

using a template as a guide. The plate was dried at 37°C, inverted with the lid open, for

approximately 1 hour. Using the template, 40 µL of standard, internal control or specimen

were placed into the appropriate wells (at room temperature). The drug was allowed to pre-

diffuse until all the wells appear completely dry and the plate was incubated at 37ºC

overnight (approximately 18 hours). The diameters of the zones of inhibition around each

well were measured using calipers (to the nearest 0.01 mm) and recorded on the results

template. Concentrations in samples were determined by placement on the standard curve

graph.

Laboratory 10: The approach was adapted from Steinmann, et al. (Mycoses. 2011;54:e421-

8). Briefly, commercially available isavuconazole 200 mg (Basilea, Basel, Switzerland) was

reconstituted in 20 mL of dimethyl sulfoxide (Roth, Karlsruhe, Germany). From this, further

dilutions were made in bovine serum (Oxoid, Wesel, Germany) to get a stock solution

containing 10 mg l-1 isavuconazole. Immediately after reconstitution, routine daily calibration

standard samples containing 0.5, 1.0, 2.0, 4.0 or 8.0 mg/L isavuconazole were prepared by

diluting the stock solution in bovine serum. The samples were stored in small portions at

–20°C. The biological activity of isavuconazole in serum samples was measured by a disc

diffusion assay. The agar medium contained 7 g yeast nitrogen base (Difco; Becton

Dickinson, Heidelberg, Germany), 7 g tryptic peptone (Merck KGaA, Darmstadt, Germany),

32

15 g glucose and 20 g agar-agar (Merck) and phosphate buffer. For phosphate buffer

production, 1.43 g KH2PO2, 10 g Na2HPO4 and 2 H2O were dissolved in 1000 mL Aqua dem.

with a final pH of 7.5. The ingredients were dispersed in the buffer and autoclaved for 15

minutes at 121°C. After being cooled to 50°C, 70 mL of the agar was dispensed into sterile

150 × 200 mm Petri dishes which were stored in plastic bags at 4°C for as long as 7 days. An

isavuconazole-hypersusceptible Candida kefyr strain (internal identifier number R34) served

as the test organism. Subcultures were performed weekly on Sabouraud dextrose agar

plates (Becton Dickinson, Franklin Lakes, NJ, USA). One colony forming unit of the

subcultured test organism was tipped and suspended in 5 mL of sterile 0.85% sodium

chloride solution; the turbidity was adjusted to a McFarland standard of 1.0. Then 200 µL of

this suspension was spread on the surface of the test plate. Paper discs (Becton Dickinson)

6 mm in diameter were impregnated with 40 µL of a standard sample, a blank control

sample, or patient sera. The loaded samples were allowed to prediffuse for 20–30 minutes at

room temperature. Zones of inhibited growth were measured after the plates had been

incubated at 35°C for 18–20 hours. The vertical and horizontal inhibition zones were

measured using an electronic vernier caliper (0–150 mm/0–6”, Insize, Zamudio [Bizkaia],

Spain). The diameter of zones of inhibited growth was plotted on a semilogarithmic graph

and the isavuconazole concentrations were placed on the logarithmic abscissa. The graph

was interpreted by estimating the isavuconazole concentration in the patient specimen and in

the internal standard sample. All test runs were performed in duplicate and for each sample,

one standard curve was prepared.

33

List of participating investigators (alphabetical by country and last name)

Austria: Robert Krause, Andreas Meinitzer (Graz)

Denmark: Rene Jörgensen (Copenhagen)

France: Delphine Allorge (Lille); Youssef Bennis (Amiens); Guillaume Deslandes (Nantes);

Marie-Claude Gagnieu (Lyon); Nicolas Gambier (Nancy); Benjamin Hennart (Lille);

Vincent Jullien (Paris); Veronique Kemmel (Strasbourg); Castaing Nadège (Bordeaux);

Damien Richard (Clermont-Ferrand); Julien Scala-Bertola (Nancy); Françoise Stanke

(Grenoble); Nicolas Venisse (Poitiers)

Germany: Carsten Müller (Cologne); Jörg Steinmann (Essen); Stefan Tönnes (Frankfurt);

Martin Wiesen (Cologne)

Italy: Jessica Biasizzo (Udine); Giuliana Congemi (Genova); Antonio D’Avolio (Torino);

Antonello Di Paolo (Pisa); Elisa Furfaro (Genova); Arianna Loregian (Padova);

Mariadelfina Molinario (Pavia); Andrea Novelli (Firenze); Federico Pea (Udine); Tanja

Zamparo (Udine)

Netherlands: Roger Brüggeman (Nijmegen)

United Kingdom: Debbie Oliver (Bristol); Caroline Moore, Malcolm Richardson, Cheryl

Wilkinson (Manchester)

34