Journal of Chromatography B 1152 (2020) 122201

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Preparation of magnetic metal organic framework and development of solid T

phase extraction method for simultaneous determination of fluconazole and
voriconazole in rat plasma samples by HPLC
Kamran Bashir, Guoning Chen, Jili Han, Hua Shu, Xia Cui, Lu Wang, Wen Li, Qiang Fu
⁎

School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, PR China

ARTICLE INFO ABSTRACT

Keywords: Fluconazole and voriconazole are the two broad-spectrum triazole antifungals. The present work described the
Fluconazole fabrication method for the synthesis of the amino-modified magnetic metal–organic framework. This material
Voriconazole was applied as a pre-sample treatment sorbent for the selective extraction of fluconazole and voriconazole in rat
Metal-organic framework plasma samples. The material was fabricated by the chemical bonding approach method and was characterized
Solid phase extraction
by different parameters. The factors which affect the extraction efficiency of the sorbent material were also
HPLC
optimized in this study. Due to the optimization of solid-phase extraction conditions, the nonspecific interaction
was reduced and the extraction recoveries of target drugs were increased in plasma samples. The extraction
method was combined with the HPLC-UV method for the analysis. Excellent linearity (0.1–25 µg/mL), detections
(0.02, 0.03 µg/mL) and quantification limits (0.04, 0.05 µg/mL) were resulted for fluconazole and voriconazole
respectively. The maximum recoveries from spiked plasma samples of fluconazole and voriconazole were 86.8%
and 78.6% and relative standard deviation were 0.9–2.8% and 2.2–3.6% respectively. Moreover, this sorbent
material was used multiple times which was an improvement over single-use commercial sorbent materials. This
validated method has practical potential for the simultaneous determination of these drugs in therapeutic drug
monitoring studies as well as for routine pharmacokinetic evaluations.

1. Introduction reported HPLC analytical method based on liquid extraction or solid-

Triazoles are derivatives of azole with a wider, safer, and less toxic
profile than imidazoles class of antifungals. Fluconazole (FZ) and vor-
iconazole (VZ) are two important members of the triazole class and
most frequently used for treating major fungal infections [1]. Despite
their clinical effects, there were certain side effects and toxicities as-
sociated with the continuous use of these drugs [2,3]. Therefore,
therapeutic drug monitoring to analyze the efficacy and toxicity of
these drugs is desired.
LC-MS or LC-MS/MS were previously reported analytical methods
for triazole drug detection [4–6]. LC-MS/MS systems are expensive,
more sophisticated, and not always available in hospital laboratories for
routine drug analysis and therapeutic drug monitoring. Due to these
reasons, HPLC is a preferred and commonly used instrument. Previously
phase extraction (SPE) [7–9]. Many microextraction and advanced ex-
traction methods were also reported for the analysis of triazole drugs in
different samples matrix such as food samples, and water samples
[10–12]. Campestre and his colleagues detected the imidazoles and
triazole drugs by HPLC- DAD in plasma samples by microextraction
packed sorbent method [13]. They used non-selective C-18 as a packed
sorbent for the extraction of VZ in plasma samples. This micro-extrac-
tion method provided good detection of analytes but the sorbent used
was not selective. So, for the therapeutic drug monitoring and phar-
macokinetic evaluation, a simple, rapid, selective, and cost-effective
analytical method is required.
Metal-organic framework (MOF) is an emerging advanced hybrid
material. MOF offered excellent features like high surface area, small
pore size, and modifiable functionality. Due to these features, MOFs are

Abbreviations: APTES, 3-Aminopropyltriethoxysilane; FZ, Fluconazole; MMOF-5, LLE, Liquid-liquid extraction; MMOF-5, Magnetic metal organic framework-5;
MSPE, Magnetic solid phase extraction; MOF, Metal organic framework, NPs, Nanoparticles; NPs, Nanoparticles; SEM, Scanning electron microscope; SPE, Solid
phase extraction; TEM, Transmission electron microscope; TEOS, Tetraethyloxisilicate; TGA, Thermo gravimetric analysis; RSD, Relative standard deviation; VZ,
Voriconazole.
⁎
Corresponding author.
E-mail address: fuqiang@mail.xjtu.edu.cn (Q. Fu).

https://doi.org/10.1016/j.jchromb.2020.122201
Received 16 March 2020; Received in revised form 19 May 2020; Accepted 29 May 2020
Available online 11 June 2020
1570-0232/ © 2020 Elsevier B.V. All rights reserved.
K. Bashir, et al.
considered promising, new, and advanced materials. The distinctively
high specific surface area supported a large adsorption capacity. The
pore structure and size of MOF assisted selective adsorption of drugs
from complex samples. Furthermore, functionalization was achieved by
simple ways [14]. MOFs were applied as a sorbent in many fields such
as chromatography, on-site sampling, and for the pre-concentration of
different kinds of drugs in SPE [15]. The MOF was magnetized either by
the paramagnetic metals or by open-shell organic ligands [16]. The
MOF magnetization improved the chemical strength, reproducibility,
and stability of the hybrid material. The MOF magnetization could be
attained by conjugating the magnetic nanoparticles (NPs) with MOF
through chemical bonding. The surface characterization [17,18] func-
tionalization of MOF cavity [19–21], post-synthesis modification [22],
and surface chemistry at the liquid–solid interface [23] were employed
to magnetized the MOF. Hu and co-workers fabricated the magnetic
nanoparticles with MOF and applied this hybrid material for the ex-
traction of gibberellic acid and polycyclic aromatic hydrocarbons [24].
We have used this synthetic approach with modification for the
synthesis of a magnetic metal–organic framework (MMOF-5).
Magnetic solid-phase extraction (MSPE) is an important and no-
ticeable extraction method among the adsorbent based extraction
techniques [25,26]. In MSPE, samples were collected easily by an ex-
ternal magnet. The filtration, centrifugation, and precipitation and
conditioning steps were avoided. MSPE offered many benefits such as
easy and simple operation, fast extraction, easy to run, more reusability,
and environmentally friendly [27–28].
Pursuing our interest in the preparation of new selective sorbent
materials, we have synthesized MMOF-5 for the extraction of FZ and VZ
in plasma samples. This is the first evidence of the separation of FZ and
VZ by MMOF-5. The first time selective SPE approach was applied for
plasma sample extraction. The prepared hybrid material was morpho-
logically and chemically characterized. The selectivity and SPE factors
were also examined and optimized. Finally, the SPE-HPLC method was
applied for the pre-sample treatment and extraction of FZ and VZ in
spiked rat plasma samples.
2. Experimental
2.1. Materials and instruments
FeCl3·6H2O was purchased from Tianjin Komiou Chemical Reagent
(China). N, N′- Dimethyl formamide (DMF) was obtained from Tianjing
Chemical Reagent Factory (China). Terephthalic acid, zinc acetate, FZ,
VZ, griseofulvin, ketoconazole were procured from Aladdin Industrial
Corporation (China). Tetraethyloxisilicate (TEOS), 3-aminopropyl-
triethoxysilane (APTES) were acquired from J&K Scientific Limited.
(China). Ethylene glycol was provided by Tianjin Fuyu Fine Chemical
(China). All other chemicals and materials were purchased locally and
of analytical grade.
A Shimadzu LC-2010AHT series HPLC (Japan) was used for ana-
lysis. The mobile phase used for the analysis was the Methanol: water
(60:40, v/v). The flow rate was 1.0 mL/min, column oven was set at
30 °C and the UV detector was set at 210 nm. The injection volume used
was 10 µL. FT-IR spectra (4000 cm−1-500 cm−1) were noted in a
Thermo Nicolet Nexus 330 FT-IR spectrometer (USA). The scanning
electron microscopic (SEM) images were attained by a TM-1000
Scanning Microscope (Japan) and transmission electron microscope
(TEM) images were obtained with H-7650 Transmission electron mi-
croscope (Japan). Thermo gravimetric analysis (TGA) was done by
using a SDT-Q600 thermo gravimetric analyzer (USA) under the air
atmosphere from room temperature to 800 °C with ramp rate of 10 °C/
min. The elementary analysis performed with an EL3 elementary ana-
lyzer (Germany). A SHA-C Vapour bathing Constant Temperature
Vibrator (China) was used for shaking purposes.
2
Journal of Chromatography B 1152 (2020) 122201
2.2. Synthesis of MMOF-5
For the synthesis of MMOF-5, the first step was to synthesize mag-
netic NPs and then modified magnetic NPs with TEOS (Fe3O4@SiO2)
and APTES (Fe3O4@NH2). The synthesis of magnetic NPs were per-
formed according to the previous method with some modifications
[29]. Briefly, 5.32 g of FeCl3·6H2O added in 160 mL of ethylene glycol
and 5.32 g of PEG-4000 added in the above mixture. The mixture was
allowed to react at 60 °C for 30 min. After that 14.4 g of anhydrous
sodium acetate was added, the above mixture was allowed to react for
further 1 h under the influence of magnetic stirring. After 1 h, the
mixture was shifted to a teflon-lined autoclave tube (200 mL). The tube
was placed in a vacuum drying oven at 200 °C for 12 h. After this step,
the mixture was allowed to cool at room temperature. Through external
magnet the magnetic NPs were separated. The unreacted reagents were
separated by washing the magnetic NPs with ethanol and deionized
water. The washing was performed multiple times. After washing,
magnetic NPs were dried at 50 °C overnight. After this step, magnetic
NPs (560 mg) were added in a mixture of 300 mL of ethyl alcohol and
ultra-pure water (4:1, v/v). The mixture was stirred for 1 h under ultra-
sonication. After stirring for 1 h, the ammonium hydroxide (9.3 mL,
25 wt%) and TEOS (1.3 mL) were added one by one in the above
mixture. The mixture was magnetically stirred for 10 h at 40 °C at
500 rpm. After 10 h the magnetic NPs were collected, separated by an
external magnet, washed with ethanol, and dried at 60 °C in the oven
overnight. The Fe3O4@SiO2 were further modified with APTES ac-
cording to the previous method with some modifications [30]. Briefly,
Fe3O4@SiO2 (0.1 g) was dispersed in the solution of HCl (0.1 M). The
mixture was stirred for half-hour. The mixture was washed with ul-
trapure water to neutralize the solution. Then magnetic particles were
added in anhydrous methanol solution (40 mL). In the final step, APTES
(800 μL) was added dropwise and the mixture was refluxed at 65 °C for
24 h. The Fe3O4@NH2 was separated and washed with methanol. After
washing the Fe3O4@NH2 NPs were dried overnight at 60 °C for 12 h.
Amino modified MMOF-5 was prepared by the previous method
with modifications [24,31]. Terephthalic acid (0.50 g) was dissolved in
40 mL of DMF. The zinc acetate (1.69 g) was dissolved in 50 mL of DMF
in a separate flask. The above solution was poured slowly in the ter-
ephthalic acid solution with continuous stirring until white precipitates
were seen. Then, 0.30 g of Fe3O4@NH2 NPs were added in the above
solution and the solution was allowed for uniform dispersion under
sonication. After 30 min of mixing, the solution was shifted into the
autoclave and the temperature was set at 120 °C for 10 h. After 10 h the
mixture was cooled and separated by an external magnet. After se-
paration the final product was washed with ethanol and then placed in
the oven for overnight drying at 60 °C.
The MOF-5 particles were also prepared by the above method with-
out the addition of Fe3O4@NH2 NPs.
2.3. Drug standards and spiked plasma samples preparations
FZ and VZ stock solutions (1000 µg/mL) were prepared in me-
thanol. The working drugs standard solutions of both drugs were pre-
pared (0.1, 1, 10, 25, 50, 75, 100, 150, 200 µg/mL) by serial dilution of
stock solution with methanol–water (50:50, v/v).
The rat blood was collected from untreated rats. The blood was
collected with heparin and was centrifuged at 12000 rpm for 3 min. The
plasma was collected and stored at − 20 °C until further use. Plasma
samples were spiked with FZ and VZ working standard solutions to
yield calibration standards in plasma at a concentration of 0.1–100 µg/
mL. The plasma samples collection and treatment were performed ac-
cording to the International guidelines and with the provision approved
by the ethical committee of Xi’an Jiaotong University (certificate no.
22–9601018). The frozen plasma (blank and spiked) samples were
thawed at room temperature before use.
K. Bashir, et al.
2.4. Selectivity experiment
In order to determine the selectivity of MMOF-5, MOF-5, and
Fe3O4@NH2, 10 mg of each sorbent material was added in respective
10 mL of each drug solution (100 µg/mL) of FZ, VZ, griseofulvin, and
ketoconazole. The flasks allowed mixing on the shaking mixer for 1 h.
After 1 h, the drug solution separated by applying external magnet and
adsorption amounts were determined by the equation (1) [32].
Q = (Co Cf ) × V/m
In this equation ‘Q’ was the adsorption capacity (µg/mg), ‘Co’, was
the Initial concentration (µg/mL), ‘Cf’ was the concentration after ad-
sorption (µg/mL), ‘V’, was the volume of sample, while ‘m’, was the
amount of the adsorbents (mg).
2.5. SPE procedure
The SPE procedure for extraction of FZ and VZ was as followed:
MMOF-5 was packed in 3 mL SPE cartridge. The cartridge was condi-
tioned with acetonitrile before packing. After the packing of the ma-
terial the sample (500 µL) was loaded and run through the column and
collected in a tube. The column was attached with vacuum apparatus
for fast and efficient run over the column. After this step, the washing of
the SPE column was done. After washing the next step is the elution.
The elution solvent was allowed to run through the SPE column and
was collected. The elution solvent was fully evaporated at room tem-
perature with the help of the nitrogen stream. After complete eva-
poration, 1 mL of methanol was added and 10 μL was injected into
HPLC, and recoveries were determined. The schematic representation
of SPE procedure was elaborated in Fig. 1.
3. Results and discussion
3.1. Synthesis and Optimization of MMOF-5
In our experiment we have used a different preparation and
Fig. 1. Schematic presentation of SPE procedure.
Journal of Chromatography B 1152 (2020) 122201
modification method for magnetic NPs and then used these particles to
fabricate the MOF-5 by previous chemical bonding method. The cova-
lent bond formation between the amino groups of modified magnetic
particles with carboxyl groups of MOF-5 particles was very necessary
for the synthesis of MMOF-5. If the Fe3O4 NPs were not amino group
modified, the material was simply a mixture of the two phases The
surface modification and content of magnetic particles are important
parameters for the covalent bond formations [24]. So, we used a dif-
ferent preparation method of magnetic NPs and also optimized the
(1)
amount of magnetic NPs (Table. S1). The prepared MMOF-5 has shown
the same characteristics and functions as early reported method with
fewer amounts of magnetic NPs used and a more easy method for the
synthesis of Fe3O4@NH2 NPs.
3.2. Characterization of MMOF-5
The morphological evaluation of MMOF-5 was performed by SEM
and TEM figures. The SEM images showed that the crystal structure of
MOF-5 was not disturbed by the chemical reaction and cubic shape
retained after the modification [31] (Fig. 2A). The TEM images verified
that the MOF-5 and Fe3O4@NH2 formed a composite material [24]
(Fig. 2B).
The FT-IR spectra (Fig. 2C) demonstrated the chemical structure of
Fe3O4@NH2, MOF-5, and MMOF-5. The C = O (−COOH) at
1705 cm−1 which was the characteristic band of the carboxyl group in
MOF-5 was not visible in the spectra of MMOF-5. This was the evidence
of a chemical reaction of carboxylate groups on the MOF-5 surface
occurred. A new band at 1685 cm−1 which was the representative band
of acylamide that appeared in the spectra of MMOF-5 represented the
formation of para-acylamino group. According to MMOF-5 spectra, a
conjugated material was formed by the combination of Fe3O4@NH2 and
MOF-5 [24].
The thermal stabilities of MOF-5, MMOF-5, and Fe3O4@NH2 were
assessed by TGA curves (Fig. 2D). The single curve showed that only
10% of the weight was lost under 400 °C which meant that composed
material has a certain and better thermal stability. Meanwhile, during
3
K. Bashir, et al.
Fig. 2. Characterization of MMOF-5. (A): SEM of MMOF-5; (B): TEM of MMOF-5; (C): FT-IR; (D): TGA.
the range of 400–500 °C, the MMOF-5 decomposed quickly. After
500 °C, the weight loss was 30%, and no further significant weight lost
observed up till 800 °C. Our prepared material exhibited excellent
thermal stability as compared to the hybrid magnetic MOF-5 [24].
According to the results of the elemental analysis the contents of
nitrogen, carbon, and hydrogen element in the MMOF-5 increased as
compared with the elemental composition of Fe3O4@NH2, MOF-5
(Table. S2). These results further confirmed the modification and
synthesis of hybrid MMOF-5.
3.3. Selectivity behavior of MMOF-5
To evaluate the selectivity, the designed material was compared
with adsorption amount of Fe3O4@NH2 and MOF-5 and executed by
using therapeutic analogues i.e; griseofulvin and ketoconazole. FZ and
VZ have a very similar chemical structure. According to these results
(Fig. 3) the MMOF-5 has more selectivity and adsorption for FZ and VZ
as compared with Fe3O4@NH2 and MOF-5. The possible mechanism of
drug adsorption was either due to π − π stacking of FZ and VZ with
MMOF-5 material or due to hydrogen bonding [33]. For griseofulvin
and itraconazole, the adsorption amount of MMOF-5 was less. Some
little adsorption was due to the π − π interaction of griseofulvin and
ketoconazole with MMOF-5 but both drugs had no hydroxyl group in
their structures. The lack of hydroxyl group made them incapable to
bind with MMOF-5 and resulted in less amount of adsorption by MMOF-
5. This investigation confirmed that the MMOF-5 has selectivity for the
FZ and VZ.
4
Journal of Chromatography B 1152 (2020) 122201
3.4. Optimization of SPE procedure
To achieve the maximum recovery from SPE procedure, we have
optimized the elementary factors such as: amount of sorbent, washing
and elution solvents, and elution solvents volumes. The adsorbent
material amount is a prime factor for effective recoveries. More
amounts of materials meant more adsorption surfaces for analytes. The
MMOF-5 amount was optimized. According to the results of this ex-
periment, a 50 mg of MMOF-5 achieved maximum recoveries of ana-
lytes (Fig. 4A). Further increase in MMOF-5 amount did not get more
recovery. A 50 mg of MMOF-5 was used as an optimized amount in
further experiments.
The plasma sample is a complex mixture of proteins, hormones, and
other metabolites. These complex substances retained on sorbents and
disturbed the extraction efficiency and consequently FZ and VZ peaks.
To decline the effect of interfering compounds, a variety of washing
solvents with different polarities were optimized. The water, water, and
methanol (7:3, v/v), phosphate buffer pH = 2.5, and methanol (5:5 v/
v), phosphate buffer pH = 2.5, and methanol (9:1 v/v) were optimized
as washing solvent. A 2 mL volume of washing solvent was used in all
experiments. The relative recoveries 83.4% and 76.6% was achieved by
using 2 mL of phosphate buffer and methanol (9:1 v/v) (Fig. 4B).
Different types of elution solvents were assessed for the extraction of
FZ and VZ from plasma samples. The methanol, methanol-acetic acid
(4:1, v/v), ethanol, ethanol-acetic acid (4:1, v/v) were tested. The
maximum recovery 85.4% and 77.3% of FZ and VZ were achieved re-
spectively by only the use of methanol (Fig. 4C). The volume of elution
solvent was also optimized. The results demonstrated that by using
K. Bashir, et al. Journal of Chromatography B 1152 (2020) 122201

Fig. 3. Selectivity of MMOF-5.

2 mL volume of the elution solvent a maximum recovery was achieved
(Fig. 4D).
So, 500 µL loading sample, 50 mg of MMOF-5, 2 mL of phosphate
buffer and methanol (9:1 v/v) as washing solvent, 2 mL of methanol as
elution solvent were chosen as the optimized quantities for the SPE
procedure.
3.5. Validation of SPE-HPLC simultaneous method
The developed SPE method was combined with the HPLC-UV
method and was validated by the determination of validation factors
such as, linearity, sensitivity, precision, accuracy, detection, and
quantification limits. A calibration graph (matrix-matched calibration)

Fig. 4. Optimization of SPE. (A): Optimization of sorbent amount; (B): Optimization of washing solvent; (C): Optimization of elution solvent; (D): Optimization of
elution solvent volume.

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K. Bashir, et al. Journal of Chromatography B 1152 (2020) 122201

Table 1
Accuracy and precision of FZ and VZ in spiked plasma samples.
Plasma Spiked drug Accuracy (Recovery Precision (RSD %,
Samples Conc (µg/mL) % n = 3) n = 3)

Intra-day Inter-day Intra- Inter-day

day

FZ 0.1 85.7 84.3 1.6 2.3

1.0 86.4 85.5 0.9 1.7
10 86.8 84.6 1.2 2.8
VZ 0.1 78.2 76.4 2.2 2.8
1.0 76.3 73.3 3.2 3.6
10 78.6 75.4 3.4 3.6

constructed by plotting the values of triplicate analysis of FZ and VZ

spiked samples (0.1–25 μg/mL). Linearity was represented by correla-
tion coefficients (R2). R2 values for FZ and VZ were 0.9997 and 0.9996
respectively. The method limit of detection and limit of quantification
for FZ were 0.02 and 0.04 (Signal to noise ratio S/N = 3) and for VZ
were 0.03, 0.05 μg/mL (S/N = 10). The intra-day and inter-day ac-
Fig. 6. Chromatogram of VZ. (A): Blank plasma; (B): VZ drug standard (10 µg/
curacy were calculated as recovery percentage achieved. The accuracy
mL); (C): Recovery from spiked plasma (10 µg/mL) after SPE.
experiments were performed at three concentration levels (0.1, 1,
10 μg/mL) and each experiment was performed three times. The intra-
day and inter-day precisions were calculated in terms of relative stan- 3.7. Reusability of MMOF-5
dard deviations (RSD) percentage at three concentration levels (0.1, 1,
10 μg/mL). The RSD % of FZ was (0.9–2.8). The RSD % of VZ was The MSPE reusability was determined to assess the effectiveness of
(2.2–3.6). The MMOF-5-SPE method combined with HPLC resulted in MMOF-5 after 7 repeated experiments. The recoveries percentage even
good recoveries (78.8–86.6%) of both drugs at the three concentration after seven cycles was ≥ 70.1%. The performance of SPE was not
levels (Table. 1). compromised even after several repeated experiments (Fig. S2). These
significant results confirmed its durability and reusability over com-
mercial and single time used sorbent materials.
3.6. Application of SPE-HPLC method

The spiked plasma samples of three different concentrations (0.1, 1,
10 μg/mL) were processed through the developed SPE procedure. The
respective chromatograms of spiked FZ and VZ were obtained. Figs. 5
and 6 display the recovery chromatograms of blank plasma, the drugs
standard (10 μg/mL) of FZ and VZ, and recovery chromatogram of
10 μg/mL spiked plasma samples after SPE procedure. The disturbance
peaks were reduced after treatment with SPE and a clear chromatogram
achieved for both drugs with little interference. Due to interference
reduction the content of the drug was improved and a recovery of 86.8
and 78.6% were achieved for FZ and VZ respectively.
3.8. Comparison with previous extraction methods
The reported method presented several benefits to investigate si-
multaneously two triazole drugs in rat plasma samples. The extraction
efficiency was compared with previously reported methods [34–38].
The results (Table. 2) showed that the developed SPE method has better
or equal recoveries when compared with previous SPE reported
methods. The SPE process is more selective, cost-effective, accurate,
specific, simple, and reproducible. However, some demerits were faced
during experiments such as time-consuming optimization methods and
slow evaporation of eluents. However the easy accessibility of instru-
ments, selectivity, high recovery, cost-effectiveness, high precision, less
organic solvent use, less amount of sample and sorbent applied, and
reusability of sorbent material for multiple analysis best fitted our SPE-
HPLC approach for therapeutic drugs evaluation and pharmacokinetic
studies.

4. Conclusion

Fig. 5. Chromatograms of FZ. (A): Blank plasma; (B): FZ drug standard
(10 µg/mL); (C): Recovery from spiked plasma (10 µg/mL) after SPE.
A new SPE procedure was adapted for the selective and simulta-
neous extraction of two triazole drugs from complex plasma samples.
MMOF-5 was synthesized by a simple chemical fabrication method and
was characterized by SEM, TEM, FT-IR, TGA, and by elemental analysis.
This material was applied as SPE sorbent material for analysis of FZ and
VZ in plasma samples. The factors affecting the SPE were optimized.
The optimized SPE offered advantages of less sorbent consumption,
reduction in sample treatment time, cost of materials, and analysis
time. Additionally, this method yields excellent analytical perfor-
mances, and the material has excellent reusability potential. This
method has applications in pre-clinical studies and pharmacokinetic
testing of these drugs in real human and animal plasma samples. The
prospect of this study is the enrichment and extraction of FZ and VZ
from real plasma samples for therapeutic drug monitoring.

6
K. Bashir, et al. Journal of Chromatography B 1152 (2020) 122201

Table 2
Comparison with previous methods.
Sample Analytes Extraction Instruments Extraction recoveries % Reference

Human Plasma Fluconazole, Itraconazole, Online SPE HPLC-MS/MS 61.4 ± 5.5, 34

Posaconazole Voriconazole 64.7 ± 2.7,
65.3 ± 1.5,
159.6 ± 1.1
Human plasma Isovuconazole Voriconazole Posaconazole Fluconazole LLE HPLC-MS 91.99 35
Caspofungin fFucytosine Itraconazole 105.0
OH-itraconazole 94.78
107.3
53.83
60.16
70.27
88.77
Human plasma Fluconazole, LLE HPLC–MS (96.62–102.42) 36
Voriconazole Isavuconazole, Posaconazole (91.60–103.02)
Itraconazole (80.14–92.45)
(96.20–97.60)
(83.48–94.56)
Rat and dog Voriconazole LLE HPLC 93.2–95.4 37
plasma
Human Plasma Ketoconazole Microextraction packed sorbent HPLC-DAD 13
and Urine Terconazole
Voriconazole
Bifonazole
Clotrimazole
Ticonazole
Econazole
Butoconazole
Miconazole
Posaconazole
Ravuconazole
Itraconazole
Human plasma ketoconazole, terconazole, voriconazole, bifonazole, Fabric phase sorptive extraction HPLC-PDA 38
and Urine clotrimazole, tioconazole, econazole, butoconazole,
miconazole, posaconazole, ravuconazole, itraconazole
Human blood and Ketoconazole Ultrasound enhanced surfactant- HPLC 80–89 10
serum Econazole nitrate assisted dispersive liquid–liquid 75–97
microextraction
Rat Plasma Fluconazole MMOF-5-SPE HPLC 86.8 Current
Voriconazole 78.6 method

CRediT authorship contribution statement References

Kamran Bashir: Conceptualization, Methodology, Data curation,
Investigation, Validation. Guoning Chen: Software. Jili Han: Data
curation. Hua Shu: Visualization. Xia Cui: Formal analysis. Lu Wang:
Investigation. Wen Li: Validation. Qiang Fu: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We gratefully acknowledge the financial support by the National
Natural Science Foundations of China (Grant No 81773689, 81573391).
We are also thankful for the generous support and scholarship provided
by the China Scholarship Council to the Kamran Bashir. We are also
thankful to the Instrumental analysis center of Xian Jiaotong University
for the technical support and help in the characterization of the mate-
rial.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jchromb.2020.122201.
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