Research Paper

Simultaneous determination of seven azole antifungal drugs in serum by ultra-high pressure liquid chromatography and diode array detection V. Mistretta, N. Dubois, R. Denooz, C. Charlier Laboratory of Clinical, Forensic, Environmental and Industrial Toxicology, CHU Sart-Tilman, University of Lie`ge, Belgium Azole antifungals are a group of fungistatic agents that can be administered orally or parenterally. The determination of the concentrations of these antifungals (miconazole, fluconazole, ketoconazole, posaconazole, voriconazole, itraconazole, and its major active metabolite, hydroxy-itraconazole) in serum can be useful to adapt the doses to pharmacological ranges because of large variability in the absorption and metabolism of the drugs, multiple drug interactions, but also potential resistance or toxicity. A method was developed and validated for the simultaneous determination of these drugs in serum utilizing ultra-high pressure liquid chromatography and diode array detection (UHPLC-DAD). After a simple and rapid liquid– liquid extraction, the pre-treated sample was analysed on an UHPLC-DAD system (Waters CorporationH). The chromatographic separation was carried out on an Acquity BEH C18 column (Waters Corporation) with a gradient mode of mobile phase composed of acetonitrile and aqueous ammonium bicarbonate 10.0 M pH10. The flow rate was 0.4 ml/min and the injection volume was 5 ml. The identification wavelength varied according to the drug from 210 to 260 nm. The method was validated by the total error method approach by using an analytical validation software (eNnoval V3.0 ArlendaH). The seven azole antifungals were identified by retention time and specific UV spectra, over a 13-minute run time. All calibration curves showed good linearity (r2.0.99) in ranges considered clinically adequate. The assay was linear from 0.05 to 10 mg/l for voriconazole, posaconazole, itraconazole, hydroxy-itraconazole, and ketoconazole, from 0.3 to 10 mg/l for fluconazole, and from 0.1 to 10 mg/l for miconazole. The bias and imprecision values for intraand inter-assays were lower than 10% and than 15%, respectively. In conclusion, a simple, sensitive, and selective UHPLC-DAD method was developed and validated to determine seven azole antifungal drugs in human serum. This method is applicable to patient samples, and can be applied successfully to clinical applications and therapeutic drug monitoring. Keywords: Azole antifungal drugs, Therapeutic drug monitoring, UHPLC-DAD, Human serum, Clinical practice

Introduction There is a major increase in the prescription of antifungal drugs in the last two decades. Nosocomial infections due to opportunistic fungal pathogens are, especially in immunocompromised patients, an important cause of morbidity or mortality.1–4 In Belgium, the azole antifungal drugs available for the systemic use (oral or parenteral) are the six following compounds: voriconazole, posaconazole, itraconazole, fluconazole, ketoconazole, and miconazole. These drugs exhibit wide inter-individual variability in pharmacokinetics at similar dosing regimens, due Correspondence to: V Mistretta, Laboratory of Clinical, Forensic, Environmental and Industrial Toxicology, CHU Sart-Tilman, University of Lie`ge, Avenue de l’Hoˆpital, 1, B-4000 Lie`ge, Belgium. Email: [email protected] chu.ulg.ac.be

ß Acta Clinica Belgica 2014 DOI 10.1179/0001551213Z.00000000018

to differences in absorption, metabolism, elimination, or due to drug interactions with a concomitant medication.5–7 In order to ensure optimal effectiveness of antifungal therapy and because many clinical trials have shown a significant correlation between plasma concentration and pharmacological response, therapeutic drug monitoring is recommended for most systemic azole antifungals, like voriconazole, posaconazole, or itraconazole.8–11 The determination of the concentration of azole antifungals in serum can help the clinician to adjust the dose administered to patients, in order to avoid insufficient concentrations or overdose, to optimize treatment and to avoid as best as possible the potential emergence of antifungal drug resistance. For this purpose, a simple, sensitive and specific ultra-high pressure liquid chromatography

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and diode array detection (UHPLC-DAD) method was developed and validated to simultaneously quantify the six azole antifungal drugs and hydroxyitraconazole, the active metabolite of itraconazole in human serum. The chemical structures of all azole drugs analysed in the present assay are shown in Fig. 1. The validation process of the method applied the total error method approach by using an analytical validation software (eNnoval V3.0 ArlendaH). The validated method involved a quick and easy liquid– liquid extraction of serum. The chromatographic analysis was carried out in thirteen minutes for all analytes. Other integrated assays of azole antifungal drugs have been published,12–17 but the originality of this approach is the use of the total error method approach as analytical validation procedure. This approach is in agreement with certification organisms, such as French Society of Pharmaceutical Sciences and Techniques (SFSTP) and has the advantage of determining the measurement uncertainty of the method which is required for accreditation (ISO 17025 and ISO 15189).18–22

Materials and Methods Chemicals and reagents Calibration, validation, and internal standards Itraconazole, ketoconazole, fluconazole, and miconazole were purchased from TCI Europe N.V. (Zwijndrecht, Belgium). Posaconazole and hydroxyitraconazole were purchased from TLC PharmaChem (Vaughan, Ont., Canada) and voriconazole from VWR International (Louvain, Belgium). Azaconazole (internal standard) was purchased from Analytical-Standards (Augsburg, Germany). Chemical purity for all compounds was higher than 99%. Quality control samples Lyophilized commercial quality control samples (QC) of voriconazole, posaconazole, itraconazole, and hydroxy-itraconazole in serum were purchased from Chromsystems (Lille, France). To prepare independent QC of other azole antifungals not available in Chromsystems QC, fluconazole was purchased from Sigma-Aldrich (Bornem, Belgium), ketoconazole from LGC Standards (Molsheim, France) and

Figure 1 Chemical structures of the seven azole antifungal drugs and the internal standard used in the assay.

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miconazole from VWR International. Chemical purity for these three compounds was higher than 99%. Solvents and other reagents HPLC supra gradient acetonitrile, HPLC supra gradient methanol and diethylether were purchased from Biosolve (Valkenswaard, The Netherlands). Dichloromethane and n-amyl alcohol were purchased from Filter Service (Eupen, Belgium), tetrahydrofuran from VWR International, and hexane from ChemLab (Somme-leuze, Belgium). Anhydrous sodium carbonate and ammonium bicarbonate were purchased from VWR International and SigmaAldrich, respectively. Drug-free human serum was obtained from blood bank (Transfusion Centre, Lie`ge, Belgium) and was stored at 220uC.

Preparation of calibration and validation standards Calibration standards allowed making and choosing the best calibration curves for each antifungal drug. Validation standards allowed assessing the analytical criteria, such as trueness, precision, limits of quantification, measurement uncertainty, and accuracy. These standards were obtained by spiking serum free of drug with separate working solutions at 100 mg/l (WS1), 10 mg/l (WS2), and 1 mg/l (WS3) of each antifungal drug. These working solutions were prepared from stock solutions by dilution in methanol and were stored between 2 and 8uC. Stock solutions of voriconazole, posaconazole, fluconazole, ketoconazole, and miconazole were prepared at a concentration of 1.0 g/l each in methanol and stored between 2 and 8uC. Stock solutions of itraconazole and hydroxy-itraconazole at 1.0 g/l were prepared in a mixture of methanol/tetrahydrofuran (50 : 50, v/v) and stored at 220uC. In this way, seven calibration standards and eight validation standards were prepared at the following concentration levels: 0.10, 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 mg/l for calibration standards and 0.05, 0.15, 0.30, 0.60, 1.5, 3.0, 6.0, and 10.0 mg/l for validation standards. These concentration levels were selected in comparison to therapeutic intervals defined in the literature.5,23,24 Each calibration standard was analysed in duplicate for 3 days and each validation standard in triplicate for 3 days.

Preparation of internal standard A stock solution of the internal standard (1 g/l of azaconazole in methanol) was diluted in methanol to obtain a 20 mg/l working solution of azaconazole. Stock and working solutions of internal standard were stored between 2 and 8uC.

Preparation of quality control samples QC samples are serum samples containing a known concentration of azole drug. They are used routinely to validate series of patient samples. Two sorts of QC

Determination of azole antifungal drugs in serum by UHPLC-DAD

samples were used: commercial QC and in-house QC. When available, three levels of commercial QC (lyophilized human serum samples) were purchased for voriconazole (0.86, 2.05, and 4 mg/l), posaconazole (0.5, 0.99, and 4.26 mg/l), itraconazole (0.27, 0.53, and 1.34 mg/l), and hydroxy-itraconazole (0.36, 0.7, and 2.05 mg/l). These QC samples were reconstituted with 1.0 ml of bidistilled water before being extracted. When not available, four levels of in-house QC (0.5, 1, 3, and 6 mg/l of fluconazole, ketoconazole, and miconazole) were prepared by spiking serum free of drug with two working solutions containing 10 and 100 mg/l of fluconazole, ketoconazole, and miconazole. These working solutions were prepared by dilution in methanol of stock solutions. These stock solutions were prepared by dissolving fluconazole, ketoconazole, and miconazole in methanol at a concentration of 1 g/l.

Sample preparation In a 20-ml conical glass tube, 500 ml of 1.0 M sodium carbonate, 5.0 ml of diethyl ether/dichloromethane/ hexane/n-amyl alcohol (50 : 30 : 20 : 0.5, v/v/v/v), and 100 ml of internal standard working solution were added to 1.0 ml of serum sample (calibration and validation standards, QC or patient samples). The mixture was then shaken for 10 minutes and centrifuged at 2000 rev/min for 11 minutes, then 3.5 ml of the supernatant was transferred into a new glass tube and evaporated to dryness at 40uC under a gentle stream of nitrogen. Sample was then reconstituted with 70 ml of bidistilled water/acetonitrile (50 : 50, v/v) and was centrifuged at 10 000 rev/min for 5 minutes. The supernatant was transferred into a standard HPLC vial and injected on the UHPLCDAD.

Apparatus UHPLC experiments were performed using an Acquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with a quaternary solvent manager, a temperature-controlled autosampler, a column-heating compartment, and a tuneable photodiode array detector (PDA detector). The analytes were separated on an Acquity Ethylene Bridged Hybrid C18 column (Waters Corporation) of 15062.1 mm (1.7 mm particle size). The column temperature was set at 40uC and the autosampler was operated at 10uC. The analysis was achieved according to the following gradient elution using acetonitrile (A) and aqueous ammonium bicarbonate 10.0 M pH 10 (B) as the mobile phase: the gradient began with 35% of phase A and 65% of phase B from 0 to 8 minutes, then it continued with 80% of phase A and 20% of phase B from 9 to 10 minutes, and came back to original conditions between 11 and 13 minutes. The flow rate was 0.4 ml/min and the injection volume was

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5 ml. The chromatographic run time was 13 minutes. The maximum absorbance wavelength varied according to the azole agent from 210 to 260 nm (lvoriconazole: 255 nm; litraconazole, lhydroxy-itraconazole, and lposaconazole: 260 nm; lfluconazole, lketoconazole, lmiconazole, and linternal standard: 210 nm). Chromatographic data were collected and analysed using the Empower software (Waters Corporation).

Analytic method validation The present method was validated by the total error method approach by using the eNnoval validation software V3.0 (ArlendaH, Lie`ge, Belgium), according to the guidelines of the SFSTP, to ISO 17025, and to ISO 15189.18–22 The eNnoval validation software V3.0 was used to assess perfect conformity to requirements, such as linearity, precision, trueness, accuracy, measurement uncertainty, limits of quantification, and detection. These criteria were obtained through calibration and validation standards.

Results A chromatogram of a spiked serum sample containing 1.0 mg/l of each azole antifungal is shown in Fig. 2. All analytes of interest were eluted within 13 minutes. No endogenous interference was found at retention time of each antifungal drug in blank serum. The concept of total error method approach has been used to evaluate the following validation parameters.

Response function The response function of an analytical procedure is, within the range, the existing relationship between the response (signal, i.e. area ratio) and the analyte concentration (quantity) in the sample.19 In the

method validation, the response function was established from the measurement of calibration standards. The simplest monotonous response function is represented by the calibration curve, which is used to calculate the concentrations of samples (validation standards, QC, or patient samples).19 The regression models for calibration curves were a weighted (1/X) linear regression for voriconazole, hydroxy-itraconazole and ketoconazole; a weighted (1/X) quadratic regression for posaconazole, itraconazole and fluconazole; and a quadratic regression for miconazole. The correlation coefficients (r2) obtained were greater than 0.999 for all calibration curves.

Linearity An analytical method is considered to be linear if, within a definite range, it allows to obtain results (i.e. measured or calculated concentrations) directly proportional to the analyte concentrations (i.e. introduced concentrations or true values) in the sample.19 Linearity was tested using validation standards for concentrations ranging from 0.05 to 10.0 mg/l. The linear relationships between calculated concentrations and introduced ones for each azole antifungal are presented in Fig. 3. The assay was linear from 0.05 to 10 mg/l for voriconazole, posaconazole, itraconazole, hydroxy-itraconazole, and ketoconazole, from 0.3 to 10 mg/l for fluconazole and from 0.11 to 10 mg/l for miconazole. The correlation coefficients (r2) obtained were greater than 0.99, the curve slopes were between 0.99 and 1.02, and intercepts were between 0.004 and 0.037.

Trueness The trueness of an analytical method expresses the closeness of agreement between the mean value

Figure 2 Chromatogram of a spiked serum sample containing 1.0 mg/l of each azole antifungal. The retention times were 1.13 minutes for fluconazole (FZ), 4.22 minutes for voriconazole (VZ), 4.93 minutes for the internal standard (IS), 7.53 minutes for ketoconazole (KZ), 8.01 minutes for posaconazole (PZ), 8.15 minutes for hydroxyitraconazole (HIZ), 9.45 minutes for itraconazole (IZ), and 10.27 minutes for miconazole (MZ).

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Figure 3 Linear relationships of azole antifungals.

obtained from a series of measurements and the value that is accepted either as a conventional true value.19 In the present method, trueness was calculated from the validation standards. Expressed in terms of relative bias, trueness reflects the systematic error. It was acceptable for each antifungal as relative biases were smaller than 10%. Results are presented in Table 1.

Precision Precision gives information about random errors and was evaluated at two levels: intra-assay and interassay at each validation standard concentration level. Coefficients of variation (CVs) are presented in Table 1, they never exceed 15%.

Accuracy profiles The accuracy profiles for each drug are based on the total error of the measurements, i.e. systematic and random errors (equivalent to trueness and precision, respectively). The total error reflects the biggest errors that can be expected in most cases.19,25 The analytical method accuracy is acceptable if, in cases where the variability is the highest (i.e. when the imprecision and inaccuracy are the highest), the deviation from the true

value does not exceed 650% for concentrations lower than 0.5 mg/l and 630% for concentrations higher or equal to this value.26 These intervals correspond to the acceptance limits of the method. The tolerance intervals (or beta-expectation limits) obtained for all levels of drug concentration tested did not exceed the acceptance limits. Results are presented in Table 1. Accuracy profiles for each drug at each concentration are presented in Fig. 4. The concept of accuracy profile was also used to determine the lower limit of quantification (LLOQ) and the range over which the method can be considered as valid. The LLOQ and the upper limit of quantification (ULOQ) were defined by the intersections between the beta-expectation limits and the acceptance limits.20,25 The limit of detection (LOD) is the smallest quantity of the drug that can be detected with a specific wavelength spectrum, but not accurately quantified in the sample; it is one-third of the LLOQ. All LLOQ, ULOQ, and LOD were obtained from validation standards. For fluconazole, LOD and LLOQ were determined as 0.09 and 0.3 mg/l, respectively. For miconazole, the LOD and LLOQ were determined as 0.03 and 0.11 mg/l, respectively. For the other azole antifungals, the LOD and LLOQ

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Note: nd5not detected.

0.05 0.15 0.30 0.60 1.50 3.00 6.00 10.00 Intra-assay PRECISION 0.05 CV (%) 0.15 0.30 0.60 1.50 3.00 6.00 10.00 Inter-assay PRECISION 0.05 CV (%) 0.15 0.30 0.60 1.50 3.00 6.00 10.00 ACCURACY 0.05 Beta-expectation tolerance intervals (%) 0.15 0.30 0.60 1.50 3.00 6.00 10.00 UNCERTAINTY 0.05 Relative expanded uncertainty (%) 0.15 0.30 0.60 1.50 3.00 6.00 10.00

TRUENESS Relative bias (%)

3.71 26.14 5.49 4.28 28.00 0.72 1.38 2.13 3.17 3.52 1.52 1.55 1.12 0.87 0.41 0.37 9.11 5.12 2.63 1.55 6.98 0.96 0.43 0.37 [216.28, 23.70] [215.48, 3.21] [0.36, 10.62] [1.79, 6.76] [224.35, 8.35] [20.84, 2.27] [0.68, 2.08] [1.55, 2.71] 20.82 11.35 5.91 3.28 16.09 2.05 0.93 0.78

Target concentration (mg/l) Voriconazole 27.28 26.48 7.73 6.58 25.48 3.81 1.30 20.94 3.55 4.20 2.81 2.44 1.19 1.33 1.02 0.67 14.18 5.40 2.81 2.93 8.84 2.01 1.14 0.67 [239.63, 25.07] [216.04, 3.07] [3.31, 12.15] [1.54, 11.61] [226.29, 15.34] [0.09, 7.53] [20.56, 3.17] [22.02, 0.15] 32.58 11.88 5.92 6.39 20.38 4.47 2.45 1.43

Pozaconazole

Table 1 Trueness, precision, accuracy, and uncertainty values obtained for each azole antifungal

25.75 29.16 4.86 7.49 25.71 2.07 1.10 21.40 3.43 4.60 3.96 3.84 2.21 1.13 1.51 1.30 9.02 7.03 4.45 4.27 8.70 1.24 1.51 1.30 [225.36, 13.86] [222.24, 3.92] [22.46, 12.18] [0.52, 14.47] [225.58, 14.17] [0.01, 4.12] [21.27, 3.48] [23.45, 0.65] 20.58 15.64 9.58 9.17 19.99 2.67 3.18 2.74

Itraconazole 6.70 24.30 6.79 4.05 27.40 2.07 1.88 2.30 3.45 3.99 2.44 2.26 0.77 0.94 0.61 1.05 6.20 5.30 2.44 3.18 8.63 1.81 1.24 1.05 [25.53, 18.92] [213.63, 5.03] [2.95, 10.63] [21.79, 9.88] [227.86, 13.07] [21.57, 5.71] [20.67, 4.43] [0.61, 3.98] 13.95 11.64 5.15 7.06 19.92 4.08 2.81 2.23

nd nd 20.25 3.62 27.22 4.18 1.89 21.19 nd nd 2.04 1.67 0.99 2.05 0.93 0.90 nd nd 2.37 2.11 8.75 2.17 1.06 2.60 nd nd [24.20, 3.71] [20.08, 7.32] [227.88, 13.45] [0.70, 7.67] [0.13, 3.65] [26.89, 4.52] nd nd 5.13 4.62 20.18 4.63 2.29 5.94

Hydroxy-itraconazole Fluconazole 21.68 25.23 8.56 6.05 27.47 2.31 0.71 20.07 6.70 3.94 2.51 3.33 1.02 1.10 0.28 0.64 14.80 4.14 3.01 3.33 8.07 1.70 0.30 1.64 [232.55, 29.19] [211.84, 1.39] [3.48, 13.64] [0.80, 11.30] [226.51, 11.57] [20.87, 5.50] [0.19, 1.22] [23.60, 3.46] 33.59 8.80 6.54 7.03 18.62 3.80 0.65 3.74

Ketoconazole

3.41 23.10 7.46 7.56 24.84 4.09 1.50 21.91 5.14 3.82 4.99 3.79 2.15 1.38 0.53 1.74 33.48 12.67 4.99 3.79 6.68 1.39 1.00 1.78 [275.15, 81.97] [231.58, 25.37] [20.39, 15.31] [1.59, 13.52] [219.74, 10.06] [1.86, 6.31] [20.50, 3.51] [24.73, 0.91] 77.18 29.06 10.51 7.99 15.31 2.94 2.26 3.77

Miconazole

Mistretta et al. Determination of azole antifungal drugs in serum by UHPLC-DAD

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Determination of azole antifungal drugs in serum by UHPLC-DAD

Figure 4 Accuracy profiles of azole antifungals. The dotted lines represent the acceptance limits (±50% and ±30%), the dashed lines represent the beta-expectation tolerance interval, and the continuous line represents the estimated relative bias. The dots represent the relative error of the measured concentrations and are plotted with respect to their targeted concentration. When the tolerance intervals are included in the acceptance limits, the assay is able to quantify accurately in the concentration range tested.

were determined in serum as 0.015 and 0.05 mg/l, respectively. For all azole drugs, the ULOQ were determined as 10 mg/l.

Uncertainty assessment The uncertainty characterizes the dispersion of the values that could reasonably be attributed to the measurand. It allows providing a quantitative assessment of the total error and its components in a single value. The expanded uncertainty represents an interval around the results where the unknown true value can be observed with a confidence level of 82.5%. The relative expanded uncertainties (%) are obtained by dividing the corresponding expanded uncertainties with the corresponding introduced concentrations.25 Values for each azole drugs are presented in Table 1.

Stability The stability of azole antifungals was proved under various storage conditions in other publications.12–14,27

It was tested in serum samples before pre-treatment kept at room temperature during 7 days, at 240 and 280uC during 6 months, in serum samples after pretreatment kept at z20uC during 24 hours, and in serum samples subjected to three freeze–thaw cycles. The stability of standard solutions was also demonstrated at room temperature during 24 hours, at 220 and 240uC during 6 months.

Clinical application The newly validated method was successfully applied to perform the determination of concentrations of voriconazole, posaconazole, fluconazole, itraconazole, and its active metabolite in 124 patient samples previously collected in tubes without anticoagulant and centrifuged at 3000 rev/min for 5 minutes. Serum samples were stored at 220uC until analysis. The measured concentrations in patient samples are presented in Tables 2 and 3. Each series of patient samples was accompanied by commercial and inhouse QC samples that were serum samples containing

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Table 2 Measured concentrations of voriconazole, posaconazole, fluconazole, itraconazole, and its active metabolite in 124 patients’ samples Concentrations (mg/l) Antifungal drugs

n

Mean

Range

Therapeutic interval (mg/l)

Voriconazole Posaconazole Itraconazole Itraconazolezhydroxy-itraconazole Fluconazole

62 52 6 6 14

2.37 0.64 0.24 0.56 2.3

0.11–10.35 0.20–1.98 0.11–0.38 0.28–0.87 0.30–3.77

1–6 1–6 0.25–2 .1 0.5–6

Table 3 Assay results of patients treated by voriconazole and posaconazole Voriconazole VfendH (n562)

Posaconazole NoxafilH (n552)

Subtherapeutic concentrations (,1 mg/l)

24%

85%

Therapeutic concentrations (1–6 mg/l)

73%

15%

Overdoses (.6 mg/l)

3%

0%

a known concentration of antifungal drugs. The QC concentrations were within 10% of the expected concentrations. Patient results revealed that 24% and 85% of patients treated by voriconazole and posaconazole, respectively, had insufficient plasma concentrations (Table 3).

Discussion An analytical UHPLC-DAD method for the quantitative determination in human serum of seven azole antifungals in the same run has been developed and validated. To our knowledge, two HPLC-DAD,14–17 one UHPLC-UV,12 two HPLC-MS/MS,13,15 and one UHPLC-MS16 assays had been published on the determination at the same time of several azole antifungals, but none has used the total error method approach, which is in agreement with certification organisms like SFSTP. This analytical validation procedure is an original validation approach using accuracy profiles based on beta-expectation tolerance intervals for the total error measurement, and assessing the measurement uncertainty that required by certification organisms, ISO 15189, and ISO 17025.21,22 In the present method, the sample pre-treatment by liquid–liquid extraction was simple, rapid, and cheaper than solid phase extraction. The analysis time was 13 minutes. The method was specific and was successfully validated by the total error method approach applied by the eNnoval software (Arlenda). The validation results indicated that the method is linear and gives accurate and reliable results for serum samples between 0.05 and 10 mg/l for voriconazole, posaconazole, itraconazole, hydroxy-itraconazole, and ketoconazole, between 0.3 and 10 mg/l for fluconazole, and between 0.11

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Outcomes Increase of hospitalization duration, medical complications, increased costs Therapeutic efficacy with a minimum time of hospitalization and a minimum cost Toxic effects: gastrointestinal disorders, headache, rash, hepatic toxicity, …

and 10 mg/l for miconazole. These dosage ranges are in agreement with the therapeutic ranges of each azole antifungal which are recommended in the literature.5,23,24 The precision and accuracy of the method are similar to those obtained in other methods.12,14 The measurement uncertainty of the present method was determined and is acceptable. In the literature, it has been reported that 20% of patients treated with recommended doses of voriconazole show subtherapeutic levels.28 Assays on patient samples achieved in this work have confirmed this observation. These preliminary results indicate the importance of the blood determination of azole antifungal drugs for cost-effective treatment of patients.

Conclusion We have developed a simple, sensitive, and specific UHPLC-DAD method to determine and quantify seven azole antifungals in human serum in a single run. These drugs are the only azole antifungals actually administered by the systemic pathway (oral or parenteral) in Belgium. This method is easy to apply to patient samples and is suitable for clinical applications, such as therapeutic drug monitoring, but also in the case of intoxication or to ensure patient compliance. Since this method has been implemented, confrontation of laboratory results and clinical data has demonstrated the value of determination of the azole antifungal drugs in blood, especially for voriconazole and posaconazole.

Acknowledgements We thank the Fonds d’Investissement pour la Recherche Scientifique from University Hospital of Lie`ge, Belgium for funding of this study (no. 4746).

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Determination of azole antifungal drugs in serum by UHPLC-DAD

quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin and caspofungin. Antimicrob Agents Chemother. 2010;54:5303–15. Ng TK, Chan RC, Adeyemi-Doro FA, Cheung SW, Cheng AF. Rapid high performance liquid chromatographic assay for antifungal agents in human sera. J Antimicrob Chemother. 1996;37:465–72. Hubert P, Nguyen-Huu, JJ, Boulanger B, Chapuzet E, Chiap P, Cohen N, et al. Harmonization of strategies for the validation of quantitative analytical procedures: a SFSTP proposal — part I. J Pharm Biomed Anal. 2004;36:579–86. Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Chiap P, Cohen N, et al. Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal — part II. J Pharm Biomed Anal. 2007;45:70–81. Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Cohen N, Compagnon PA, et al. Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal — part III. J Pharm Biomed Anal. 2007;45:82–96. International Organization for Standardization. General requirements for the competence of testing and calibration laboratories. ISO/IEC 17025. Geneva: International Organization for Standardization; 2005. International Organization for Standardization. Medical laboratories — particular requirements for quality and competence. ISO 15189. Geneva: International Organization for Standardization; 2012. Hulin A, Deguillaume AM, Bretagne S, Be´zie Y. Bon usage des antifongiques dans le traitement des candidoses et aspergilloses invasives. J Pharm Clin. 2005;24:125–38. Schulz M, Schmoldt A. Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics. Die Pharm. 2003;58:447–74. Rozet E, Ceccato A, Hubert C, Ziemons E, Oprean R, Rudaz S, et al. Analysis of recent pharmaceutical regulatory documents on analytical method validation. J Chromatogr A. 2007;1158:111–25. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharm Res. 2007;24:1962–73. Kobylin´ska M, Kobylin´ska K, Sobik B. High-performance liquid chromatographic analysis for the determination of miconazole in human plasma using solid-phase extraction. J Chromatogr B. 1996;685:191–5. Sanford JP, Gilbert DN, Chambers HF, Eliopoulos GM, Moellering RC, Saag MS. The Sanford guide to antimicrobial therapy 2010–2011. Belgian/Luxembourg edition. Bruxelles: SBIMC-BVIKM; 2010. p. 213.

Acta Clinica Belgica

2014

VOL .

69

NO .

1

61

Simultaneous determination of seven azole antifungal drugs in serum by ultra-high pressure liquid chromatography and diode array detection.

Azole antifungals are a group of fungistatic agents that can be administered orally or parenterally. The determination of the concentrations of these ...
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