Analysis of imidazoles and triazoles in biological samples after MicroExtraction by packed sorbent

Figures & data

Figure 1. Chemical structures of the selected 12 azoles.

Figure 2. Schematic illustration of the optimized MEPS extraction procedure.

Figure 3. Chromatograms obtained after the extraction and analysis of 12 azoles and benzyl-4-hydroxybenzoate (IS) at the wavelength of 210 nm, respectively (up, plasma sample and, down, urine sample: (a) blank sample, (b) blank sample spiked with 5 μg mL⁻¹ of IS and (c) blank sample spiked with 5 μg mL⁻¹ of IS and 4 μg mL⁻¹ of different drugs). 20 μL of samples were injected during the analysis.

Table 1. Mean linear calibration curve parameters performed by weighted-linear least-squares regression analysis of six independent eight non-zero concentration points in plasma samples.

	Linearity range	Slope^a		Intercept^a		Determination	Wavelength	Retention time (min)
		Analyte	(μg mL^−1)	Mean	Std. dev.			Mean	Std. dev.	coefficient (r^2)	(nm)	Mean	Std. dev.
Ketoconazole	0.05–5 (0.017 μg mL^−1)^b	0.07762	±0.00485	−0.00211	±0.00019	0.9846		5.49	±0.12
Terconazole	0.2–5 (0.070 μg mL^−1)^b	0.1906	±0.016364	0.01732	±0.00207	0.9954		6.63	±0.14
Voriconazole	0.05–5 (0.017 μg mL^−1)^b	0.084313	±0.00568	0.002999	±0.002959	0.9917		9.94	±0.13
Bifonazole	0.05–5 (0.017 μg mL^−1)^b	0.376833	±0.036575	0.01551	±0.002246	0.9910		11.23	±0.30
Clotrimazole	0.05–5 (0.017 μg mL^−1)^b	0.1885	±0.015932	0.01477	±0.001127	0.9961		12.75	±0.45
Tioconazole	0.05–5 (0.017 μg mL^−1)^b	0.113067	±0.014298	0.005134	±0.000549	0.9908	210	14.19	±0.34
Econazole	0.05–5 (0.017 μg mL^−1)^b	0.129033	±0.014347	0.003792	±0.002456	0.9927		17.84	±0.45
Butoconazole	0.05–5 (0.017 μg mL^−1)^b	0.2452	±0.022889	0.002835	±0.004686	0.9925		22.64	±1.84
Miconazole	0.05–5 (0.017 μg mL^−1)^b	0.129033	±0.020994	−0.00083	±0.003097	0.9922		23.22	±0.12
Posaconazole	0.05–5 (0.017 μg mL^−1)^b	0.1483	±0.021392	−0.00155	±0.001385	0.9962		24.01	±0.09
Ravuconazole	0.02–5 (0.007 μg mL^−1)^b	0.032007	±0.002906	0.002364	±0.000659	0.9902		26.06	±0.11
Itraconazole	0.05–5 (0.017 μg mL^−1)^b	0.059923	±0.011036	0.015467	±0.002012	0.9924		28.31	±0.10

^a Values at 95% confidence intervals on the mean of six independent calibration curves.

^b The round brackets show the LOD values obtained from signal-to-noise ratio³; the slope and intercept of calibration curve are expressed in μg mL⁻¹.

Table 2. Mean linear calibration curve parameters performed by weighted-linear least-squares regression analysis of six independent eight non-zero concentration points in urine samples.

	Linearity range	Slope^a		Intercept^a		Determination	Wavelength	Retention time (min)
		Analyte	(μg mL^−1)	Mean	Std. dev.			Mean	Std. dev.	coefficient (r^2)	(nm)	Mean	Std. dev.
Ketoconazole	0.05–5 (0.017 μg mL^−1)^b	0.093827	±0.005462	−0.00211	±0.00019	0.9877		5.49	±0.12
Terconazole	0.2–5 (0.070 μg mL^−1)^b	0.182667	±0.019981	0.01732	±0.00207	0.9938		6.63	±0.14
Voriconazole	0.05–5 (0.017 μg mL^−1)^b	0.070917	±0.001684	0.002999	±0.002959	0.9934		9.94	±0.13
Bifonazole	0.05–5 (0.017 μg mL^−1)^b	0.384167	±0.082413	0.01551	±0.002246	0.9958		11.23	±0.30
Clotrimazole	0.05–5 (0.017 μg mL^−1)^b	0.199733	±0.044602	0.01477	±0.001127	0.9970		12.75	±0.45
Tioconazole	0.05–5 (0.017 μg mL^−1)^b	0.1181	±0.018455	0.005134	±0.000549	0.9901	210	14.19	±0.34
Econazole	0.05–5 (0.017 μg mL^−1)^b	0.135533	±0.0252	0.003792	±0.002456	0.9870		17.84	±0.45
Butoconazole	0.05–5 (0.017 μg mL^−1)^b	0.232067	±0.01817	0.002835	±0.004686	0.9944		22.64	±1.84
Miconazole	0.05–5 (0.017 μg mL^−1)^b	0.134333	±0.019278	−0.00083	±0.003097	0.9980		23.22	±0.12
Posaconazole	0.05–5 (0.017 μg mL^−1)^b	0.2156	±0.020832	−0.00155	±0.001385	0.9951		24.01	±0.09
Ravuconazole	0.02–5 (0.007 μg mL^−1)^b	0.03004	±0.003518	0.002364	±0.000659	0.9969		26.06	±0.11
Itraconazole	0.05–5 (0.017 μg mL^−1)^b	0.098023	±0.011686	0.015467	±0.002012	0.9944		28.31	±0.10

^a Values at 95% confidence intervals on the mean of six independent calibration curves.

^b The round brackets show the LOD values obtained from signal-to-noise ratio³; the slope and intercept of calibration curve are expressed in μg mL⁻¹.

Figure 4. Effects of cycles number on process efficiency for the different analytes during sample loading and sample elution. Values are reported as percentage of observed analyte area respect to a water-based sample spiked at the same concentration level and submitted to the same extraction procedure.

Table 3. Comparison of published analytical methods for the analysis of azoles extracted from different biological samples.

Sample	Analytes	Extraction	Instrument setting up	Stationary phase	Total extraction procedure time (minutes)	Chromatographic analysis (minutes)	LOQ (μg mL^−1)	Linear dynamic range (μg mL^−1)	Ref.
Serum	Posaconazole	Column switching	HPLC-FLD	C18	3	17	0.1	0.1–5	^Citation13
Plasma/saliva	Voriconazole	LLE	HPLC-FLD	C18	6	12	0.1	0.1–10	^Citation12
Plasma	Itraconazole/metabolite	LLE	HPLC-FLD	C18	25	30	0.005	0.005–0.5	^Citation11
Plasma	Itraconazole	Protein precipitation	HPLC-UV/Vis	C18	10	–	0.08	–	^Citation14
Plasma	Voriconazole	LLE	HPLC-UV/Vis	C18	15, plus dryness	20	0.1	0.1–20	^Citation15
	Posaconazole						0.05	0.05–10
Plasma	Posaconazole	–	HPLC-PDA	C8	–	65	–	–	^Citation16
Plasma	Itraconazole	Protein precipitation	HPLC-MS/MS	C18	–	–	0.001	0.001–0.5	^Citation20
Plasma	Voriconazole	LLE	HPLC-MS/MS	C18	12	–	0.027	–	^Citation21
Plasma	Posaconazole	LLE	HPLC-MS/MS	C18	–	–	0.001	–	^Citation22
Interlaboratory Test	Itraconazole	–	–	–	–	–	–	–	^Citation23
	Voriconazole
	Posaconazole
	Fluconazole
Formulation	Itraconazole	–	HPLC-UV/Vis	Sb-Aq	10	–	–	–	^Citation24
Formulation	Voriconazole	–	HPLC-UV/Vis	C18	–	20	–	–	^Citation25
	Related impurities
Ophtalmic	Voriconazole	–	HPLC-UV/Vis	C18	–	–	–	–	^Citation26
Plasma	Voriconazole	LLE	HPLC-UV/Vis	C8	25, plus dryness	20	0.2	0.2–10	^Citation27
	Posaconazole						0.05	0.05–10
Plasma	Fluconazole	SPE	HPLC-UV/Vis	C6-phenyl	–	19	0.05	0.05–50	^Citation30
	Posaconazole							0.05–40
	Voriconazole							0.05–40
	Itraconazole/metabolite							0.05–40
Cosmetic	Ketoconazole	Ultrasound and Liquid Extraction	HPLC-UV/Vis	RP amide C16	10, plus centrifugation	80	0.05	0.05–100	^Citation28
	Tioconazole
	Econazole
	Miconazole
	Itraconazole
	Clotrimazole
Plasma	Itraconazole	Protein precipitation	HPLC-MS	C18	10	13	0.03	0.03–8	^Citation19
	Voriconazole						0.03	0.03–8
	Posaconazole						0.04	0.04–10
Plasma	Itraconazole	Protein precipitation	HPLC-UV/Vis	C6-phenyl	5	17	0.05	0.05–10	^Citation17
	Voriconazole
	Posaconazole
Rat plasma	Itraconazole	LLE	HPLC-DAD	C18	12, plus dryness	–	0.05	0.05–5	^Citation18
	Posaconazole
Plasma	Posaconazole	Protein precipitation	HPLC-MS/MS	C18 and PFP	10	4	0.1	0.1–20	^Citation42
	Voriconazole
	Itraconazole/metabolite
Plasma	Voriconazole	LLE	UPLC-UV/Vis	BEH Phenyl	10, plus dryness	6	0.05	0.05–10	^Citation43
	Posaconazole
	Isavuconazole
	Itraconazole/metabolite
Plasma	Itraconazole	SLE	UHPLC-MS/MS	Kinetex F5 (PFP)	34	7.6	0.005	0.005–2.5	^Citation39
	Hydroxy itraconazole
	Keto itraconazole
	N-Desalkyl itraconazole
Serum	Voriconazole	Protein precipitation	UFLC-MS/MS	Kinetex C18	6	4	0.1	0.1–10	^Citation44
Plasma/urine	Ketoconazole	MEPS	HPLC-DAD	C18	7	36	0.05	0.05–5	Current paper
	Terconazole						0.2	0.2–5
	Voriconazole						0.05	0.05–5
	Bifonazole						0.05	0.05–5
	Clotrimazole						0.05	0.05–5
	Tioconazole						0.05	0.05–5
	Econazole						0.05	0.05–5
	Butoconazole						0.05	0.05–5
	Miconazole						0.05	0.05–5
	Posaconazole						0.05	0.05–5
	Ravuconazole						0.02	0.02–5
	Itraconazole						0.05	0.05–5

PFP: pentafluorophenyl; RP: reversed phase; LLE: liquid–liquid extraction; SLE: solid-supported liquid extraction; plus dryness: required time for complete solvent evaporation is not reported; plus centrifugation: required time for sample centrifugation is not reported.

Table 4. Quantitative analysis of plasma or urine samples collected from healthy human volunteers after single oral dose of commercial capsules of itraconazole (100 mg) and commercial tablets of miconazole (500 mg). Plasma was collected 4 h after the oral administration of drugs, while urine samples were collected at different times.

						Itraconazole^c		Miconazole^c
						Matrix	Sample no.	Formulation	Dose (mg × 2/die)	Time (h)^a	Volume (mL)	Concentration (μg mL^−1)	Total amount (mg)	Concentration (μg mL^−1)	Total amount (mg)
Plasma	1	Capsules	100	4	4400^b							0.124 (±0.07)	0.546	n.d.	n.d.
	2	Tablets	500	4	4400^b	n.d.	n.d.	0.508 (±0.05)	2.235
Urine	3	Capsules	100	0	100^d	n.d.	n.d.	n.d.	n.d.
	4	Capsules	100	3	110^d	n.d.	n.d.	n.d.	n.d.
	5	Capsules	100	6	100^d	n.d.	n.d.	n.d.	n.d.
	6	Capsules	100	9	110^d	n.d.	n.d.	n.d.	n.d.
	7	Capsules	100	12	110^d	n.d.	n.d.	n.d.	n.d.
	8	Capsules	100	18	110^d	0.402 (±0.09)	0.044	n.d.	n.d.
	9	Capsules	100	24	110^d	n.d.	n.d.	n.d.	n.d.
	10	Capsules	100	36	110^d	n.d.	n.d.	n.d.	n.d.
	11	Tablets	500	0	80^d	n.d.	n.d.	n.d.	n.d.
	12	Tablets	500	3	380^d	n.d.	n.d.	n.d.	n.d.
	13	Tablets	500	6	140^d	n.d.	n.d.	0.136 (±0.08)	0.019
	14	Tablets	500	9	160^d	n.d.	n.d.	0.229 (±0.04)	0.037
	15	Tablets	500	12	120^d	n.d.	n.d.	0.102 (±0.07)	0.012
	16	Tablets	500	21	420^d	n.d.	n.d.	0.098 (±0.06)	0.041
	17	Tablets	500	24	40^d	n.d.	n.d.	0.048 (±0.03)	0.002
	18	Tablets	500	36	60^d	n.d.	n.d.	n.d.	n.d.

^a Time between the last drug administration and sample collection.

^b According to³⁹.

^c In round brackets are reported standard errors of the mean (SEM) obtained from three independent measures.

^d Urine volumes collected from treated patients. n.d.: not detected.

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