Microchemical Journal 172 (2022) 106906

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Homogeneous liquid phase microextraction using hydrophilic media for the

determination of fluoroquinolones in human urine using HPLC-FLD
Eleni Tsanaktsidou a, Catherine K. Markopoulou a, Paraskevas D. Tzanavaras b,
Constantinos K. Zacharis a, *
a
Laboratory of Pharmaceutical Analysis, Department of Pharmaceutical Technology, School of Pharmacy, Aristotle University of Thessaloniki, GR-54124, Greece
b
Laboratory of Analytical Chemistry, School of Chemistry, Faculty of Sciences, Aristotle University of Thessaloniki, GR-54124, Greece

A R T I C L E I N F O A B S T R A C T

Keywords: Miniaturization of liquid-phase extraction is an increasingly emerging field of sample preparation aiming to
Homogeneous liquid phase microextraction reduce the solvent consumption and to protect the environment. In this paper, a homogeneous liquid phase
Fluoroquinolones microextraction using a hydrophilic extraction media in combination with a salt as phase-forming agent was
Human urine
developed for the determination of four fluoroquinolones (ciprofloxacin, norfloxacin, moxifloxacin and pruli­
High pressure liquid chromatography
floxacin) in human urine. Important parameters affecting the extraction performance including sample volume,
Fluorescence detection
Accuracy profiles organic solvent volume, salt concentration etc were systematically investigated and optimized. Multivariate
experimental designs namely Plackett-Burman and Box-Behnken designs were sequentially applied for factor
screening and optimization of the method parameters.
The method was fully validated using the “total error” approach. Accuracy profiles – a graphical decision-
making tool – were constructed using the results of the validation procedures. The β-expectation tolerance in­
tervals did not exceed the acceptance criteria of ± 15%, meaning that 95% of future results will be included in
the defined bias limits. The relative bias ranged between ─ 2.8 to 7.7 % for all analytes, while the RSD values for
repeatability and intermediate precision were less 6.6%. The achieved limits of detection (LOD) were ranged
between 3 and 11 ng mL− 1 while the lower limits of quantitation (LLOQ) were established as 10 ng mL− 1 for
ciprofloxacin and norfloxacin and 100 ng mL− 1 for moxifloxacin and prulifloxacin, respectively. The ruggedness
of the microextraction procedure was assessed through Monte-Carlo simulations and capability analysis. The
proposed approach improves the procedures proposed to date for the bioanalysis of fluoroquinolones in terms of
efficiency, reduction of the sample volume and extraction time.

1. Introduction
Fluoroquinolones (FQs) is a group of antibiotics that exhibit strong
antimicrobial action against both gram-positive and gram-negative
pathogens (broad-spectrum antibiotics). They are classified into four
generations where ciprofloxacin (CIP) and norfloxacin (NOR) belong
to the 2nd generation while moxifloxacin (MOX) and prulifloxacin
(PRU) to the 4th generation [1]. The action mechanism of FQs is
related to the creation of a selective complex with DNAgyrase of gram
(-) bacteria and topoisomerase IV of gram(+) bacteria resulting to the
split of bacterial chromosomes [2]. The use of FQ in clinical practice is
crucial for the treatment of urinary tract infections and infections of
the digestive tract and respiratory system [1]. They exhibit very good
bioavailability (at least 70%) which is characterized by concentration-
dependent bactericidal activity but also some side effects including
tendon rupture, neurological and psychiatric symptoms have been also
reported [3,4].
In literature, several analytical methods have been reported on the
determination of FQs in urine matrixes. Among these, separation-
driven are the predominant techniques offering selective and sensi­
tive determinations [5]. Recently reported methods for the analysis of
urinary FQs include HPLC with UV [6] / fluorescence (FLD) [7], in
series UV-FLD [8] or mass spectrometric (MS) detection [9]. Due to the
complexity of the biological matrix and the low concentration levels of
drugs, sample preparation is required prior to instrumental analysis.
Traditional techniques such as liquid–liquid extraction (LLE) and solid-
phase extraction (SPE) are the most common sample pretreatment
methods for this kind of analysis. From the Green Analytical Chemistry

* Corresponding author.
E-mail address: czacharis@pharm.auth.gr (C.K. Zacharis).

https://doi.org/10.1016/j.microc.2021.106906
Received 24 August 2021; Received in revised form 23 September 2021; Accepted 6 October 2021
Available online 11 October 2021
0026-265X/© 2021 Elsevier B.V. All rights reserved.
E. Tsanaktsidou et al. Microchemical Journal 172 (2022) 106906

point of view, these techniques are considered unfavorable due to the Table 1
drawbacks mainly related to the waste of solvents, toxic reagents, Validation results for the quantitation of CIP in human urine.
consumption of time and energy. Other drawbacks include the limited Validation criteria
extraction recoveries and the relatively poor selectivity especially
Response function Slope (×10- Intercept r
when the physicochemical properties of the analytes vary over a wide (linear unweighted) 3
) (×10-3)
range [10]. (ka = 5; m = 5; n = 3) (10 ─ 100 ng mL− 1)
New trends in sample preparation comprise the use of simpler, Day 1 88.9 386.6 0.9990
automated, miniaturized and “green” analytical schemes [11]. The basic Day 2 89.2 291.1 0.9995
Day 3 84.8 433.0 0.9995
philosophy is the fulfillment of the 12 principles of Green Analytical Precision (k = 5; n = 3)
Chemistry towards the maximization of the eco-friendly character of the C (ng mL− 1) sr (%)b sR (%)c
analytical methods. Various novel microextraction techniques have 10 6.6 5.2
therefore been developed for the determination of FQ in urine involving 25 1.4 2.6
50 1.3 2.3
dispersive micro solid-phase extraction (DMSPE) [12], stir flat sheet
100 3.3 3.5
membrane liquid microextraction [13], liquid phase microextraction Trueness (k = 5; n = 3)
using microchip device [14], single drop liquid–liquid–liquid micro­ C (ng mL− 1) Relative bias (%)
extraction [15], ionic liquid-based microextraction system [16], salting- 10 + 6.6
out assisted liquid phase microextraction [17,18] and effervescence- 25 + 3.0
50 + 6.4
assisted liquid phase microextraction with switchable solvent [19]. 100 + 4.7
Homogeneous liquid phase microextraction (HLPME) has attracted Accuracy (k = 5; n = 3)
much attention in the last years [20]. The principle of this technique is C (ng mL− 1) Relative β-ЕΤІ (%)
based on the usage of a low dielectric constant hydrophilic organic 10 [3.08, 10.12]
25 [─ 6.70, 12.64]
solvent (acetonitrile, methanol, acetone, tetrahydrofuran etc), which is
50 [─ 1.82, 14.56]
completely soluble with water [21]. The contact area between the two 100 [─ 3.40, 12.77]
“phases” is principally infinite compared to other immiscible solvent-
based LLE or dispersive liquid liquid microextraction (DLLME) leading Linearity (k = 5; n = 3; m = 5)(10–100 ng
to higher mass transfer. The phase separation can be accomplished by i) mL− 1)

changing the temperature, ii) altering the pH of perfluorinated surfac­

tants, iii) the formation of ion-pair and iv) the addition of an electrolyte
(salting-out) [21,22]. In the latter case, two theories are currently pre­
sumed to describe the salting-out phenomenon. One of them claims that
the more polar solvent favorably surrounds the electrolyte (salt) due to
the Coulombic forces while an alternative theory states that one of the
solvents preferentially solvates the electrolyte (ionic or not ionic)
making it unavailable to dissolve the other solvent [23]. However, it is
possible that both phenomena can act synergistically. The main char­
acteristics of this technique comprise simplicity in operation, low cost,
reduction the extraction time and usage of non-halogenated or aromatic
solvents. Additionally, the extracts are fully compatible with reversed
phase HPLC enabling direct injection.
The purpose of the present study is the systematic development and
thorough optimization of a homogeneous liquid-phase microextraction
scheme for the determination of four FQs in human urine. Following
extraction, the analytes were processed by HPLC-FLD. Experimental de­
signs were exploited to identify the parameters that most affecting the
extraction performance and to find the optimum HLPME experimental
conditions. The described method was fully validated using the accuracy
profiles which take into consideration the total error (systematic and
random errors of the results). The proposed analytical scheme offers sig­
nificant advantages including simplicity, easy handling, cost-effectiveness
and high extraction recoveries.
2. Experimental
2.1. Reagents and solutions
Ciprofloxacin (CIP), norfloxacin (NOR), moxifloxacin (MOX) and
prulifloxacin (PRU) were obtained from commercially available phar­
maceutical formulations Ladinin (CIP solution, 400 mg/200 mL, Phar­
mathen Ltd), Vetamol (NOR, 400 mg/tab, Viofar), Microbiel (MOX
solution, 400 mg/250 mL, Cooper) and Prixina (PRU, 600 mg/tab,
Angelini Pharma Hellas). Nalidixic acid (≥99.0%) used as internal
standard was purchased from Sigma-Aldrich (St Louis, MO, USA). The
chemical properties of the analytes are tabulated in Table 1S. Acetoni­
trile, methanol (Riedel-de Haen, Honeywell Chemicals), isopropanol
(VWR Chemicals, Vienna, Austria) and acetone (Sigma-Aldrich, Stein­
heim, Germany) were all of analytical grade. Inorganic salts ZnSO4,
Slope 1.055
Intercept ─ 1.355
r2 0.9993
LOD (ng mL− 1) 3
LLOQ (ng mL− 1) 10
a
k, m and n correspond to the number of experiments, calibration levels and
replicates, respectively
b
sr (%): relative standard deviation under repeatability conditions
c
sR (%): relative standard deviation under intermediate precision
MgSO4, NaCl, KCl, Na2SO4, (NH4)2SO4, NaH2PO4, H3PO4 and trime­
thylamine (TEA) (≥99% in all cases) were provided by Merck (Darm­
stadt, Germany). High-purity water (18.2 MΩ cm resistivity) that was
used for the HPLC analysis was produced by a B30 water purification
system (Adrona SIA, Riga, Latvia).
For the optimization of HLPME parameters, artificial urine was pre­
pared according to the literature [24] by dissolving lactic acid (0.1 g),
citric acid (0.4 g), sodium bicarbonate (2.1 g), urea (10 g), CaCl2⋅2H2O
(0.37 g), NaCl (5.2 g), MgSO4⋅7H2O (0.49 g), Na2SO4⋅10H2O (3.2 g),
KH2PO4 (0.95 g), K2HPO4 (1.2 g) and NH4Cl (1.3 g) in 1 L H2O. The pH of
the final solution was adjusted to 6.0 by addition of 1.0 mol L-1 HCl.
Standard analyte and ISTD stock solutions (1000 μg mL− 1) were
prepared in methanol. All solutions were filtered and stored at 4 ◦ C
protected from the light. Working standard solutions were prepared
daily by appropriate dilutions of aliquots obtained by the stock solutions
in water. Stock solutions were routinely measured to quantify degra­
dation (a 5% difference compared to the original signals was set as
threshold). All glassware was previously washed with water and acetone
in order to avoid carry over effects.
2.2. Instrumentation and chromatographic conditions
All chromatographic separations were performed on a Shimadzu
HPLC binary solvent system (Kyoto, Japan) equipped with two LC-20AD
high pressure gradient pumps, an SIL-20AC HT autosampler, a CTO-
10ASVP column compartment and a RF-535 fluorescence detector.
The instrument control and the data handling were carried out via LC
Solutions software (version 1.25 SP4). A reversed phase Hypersil BDS
C18 column (250 × 4.6 mm, 5 μm, Thermo Scientific) was utilized for the
separation of the analytes using a binary gradient elution program using

2
E. Tsanaktsidou et al.
an aqueous solution of 25 mM H3PO4 (pH 3 adjusted with TEA) (mobile
phase A) and methanol/acetonitrile 15/85 % v/v (mobile phase B). The
initial ratio was 10 % v/v of B for 5 min and linearly increased to 30 % in
15 min, kept constant up to 28 min and then changed to the initial ratio
at 30 min and stated up to 45 min to maintain a stable and reproducible
separation. The flow rate was set to 1 mL min− 1 and the injection volume
was 20 μL. The column was thermostated to 45 ◦ C. The analytes were
detected spectrofluorimetrically at λex/λem = 280/450 nm.
A heating block (Pierce, Reacti-Therm) coupled with a nitrogen gas
generator (Claind) was used for the evaporation of the LPME extractants
to dryness. Glass tight syringes with volumes of 100 μL and 1000 μL
(Hamilton, Nevada, USA) were used for measurements of the volumes of
the extraction solvents.
2.3. Urine sampling and HLPME protocol
Urine samples were collected from healthy volunteers after written
informed ethical consent. Prior urine collection, no medication was
taken by the volunteers. Following collection, the samples were centri­
fuged at 3000 g for 5 min and the supernatants were stored in sterilized
containers at − 18 ◦ C until their analysis. For method validation, a
pooled drug-free urine sample was prepared by mixing equal volumes of
six different samples (n = 6). Prior to extraction the sample was spiked
with known amounts of the analytes and ISTD (10 μg mL− 1).
In HLPME procedure, an aliquot of 200 μL of diluted (1:1) urine
sample (blank or spiked) was transferred in 1.5 mL Eppendorf tubes.
500 μL ACN were added and the mixture was vortexed for 10 s. After­
wards, 200 μL of Na2SO4 solution (2.5 mol L-1) was added in the
aqueous-acetonitrile homogeneous phase and the mixture was vortexed
for 10 s and centrifuged at 2000 g for 5 min to promote the phase sep­
aration. The upper analyte-enriched acetonitrile phase (ca 450 μL) was
Fig. 1. Pareto charts of the main effects of the studied parameters in Plackett-Burman screening design for A) CIP, B) NOR, C) MOX and D) PRU.
3
Microchemical Journal 172 (2022) 106906
retrieved using a glass syringe and transferred to an HPLC vial. The
extractant was then evaporated to dryness using a gentle N2 flow and
finally reconstituted in 250 μL of mobile phase prior to analysis. A
flowchart of the optimum HPLME protocol is illustrated in Fig. 1S.
For the establishment of the accuracy profiles, five concentration
levels (m = 5) namely 5, 10, 25, 50 and 100 (for CIP and NOR) and 50,
100, 250, 500 and 1000 ng mL− 1 (for MOX and PRU) were prepared in
triplicate (n = 3) for each series of experiments (k = 5), respectively.
Independent stock solutions of the analytes were used for the prepara­
tion of spiked urine validation samples in order to obtain the desired
concentration level.
2.4. Accuracy profiles
The validation of the analytical method was implemented by con­
structing the accuracy profiles. This approach is based on the graphical
report of i) the acceptability intervals that describe the required per­
formance of the analytical method and ii) the β-expectation tolerance
intervals (β-ЕΤІ). The latter represents the interval where it is expected
that a proportion β of future measurements will be within the acceptance
limits λ [25]. In should be stated that the accuracy profiles could be also
informative for the realistic estimation of the limit of quantitation (LOQ)
of the method. The mathematical expression of the analytical profile is
given below:
[ )√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
(
1+β 1
bias(%)j − Qt v;
2
1+
pnB2j
sr,j ; bias(%)j
( )√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ]
1+β 1
+ Qt v; 1+ sr,j
2 pnB2j
E. Tsanaktsidou et al.
√̅̅̅̅̅̅̅̅̅̅̅̅
√ 2
√ ̂σ B,j +1
̂μ j − μTj
2 2
̂σ W,j +̂σ B,j
√ 2
√ ̂σ
where bias (%)j = μ × 100, sr,j = × 100, Bj =√ W,j2
Tj ̂μ j √ ̂σ B,j
n 2 +1
̂σ W,j
(R+1)2
R+n(p−1 1)+1− 1
pn2
-̂μ j is the estimate of the mean results of the jth concentration level
-̂μ T is the unknown “true value”
- p is the number of series
- n is the number of the independent replicate per series
- Qt (ν ;1+β
2 ) is the β quantile of the t-Student distribution with ν de­
grees of freedom
σ 2W,j is the within series variance
-̂
σ 2B,j is the between series variance
-̂
2.5. Calculation of extraction recovery
The extraction recovery (ER %) was employed for the evaluation of
the extraction efficiency of the proposed analytical scheme. This term
was estimated using the following expression:
Corg × Vorg
ER% = × 100
Caq × Vaq
where Corg and Caq are the concentrations of the analyte in the
organic and aqueous phase (1000 ng mL− 1), and Vorg and Vaq are the
volume of upper organic and aqueous layer, respectively. The calcula­
tion of Corg was performed by external standardization by direct injec­
tion of standard solutions in the range of 100 – 2000 ng mL− 1 prepared
in water.
3. Results and discussion
3.1. Optimization of the chromatographic conditions
From a HPLC point of view, due to the similarity of the chemical
structures of the analytes, (especially for the CIP/NOR pair) the
achievement of sufficient/acceptable resolution (Rs > 1.5) is chal­
lenging. Based on the reported literature, initial experimentation was
involved using a reversed phase C18 column [26,27]. The knowledge of
pKa values is particularly important. The studied FQs are amphoteric
which contain both carboxylic and amino groups with pKa1 and pKa2
values in the range of 5.69 – 6.34 and 5.16 – 9.42, respectively
(Table 1S). On this basis, the pH of the acetonitrile-containing mobile
phase was lowered below the pKa1 of the drugs by the addition of an acid
(H3PO4, TFA both 0.1% v/v). Both acids resulted in more symmetrical
peaks due to the minimization of the secondary interactions of the
analytes with the residual silanol groups of the stationary phase. Phos­
phoric acid exhibited slightly better resolution between the analytes and
higher fluorescence intensities and therefore was adopted for subse­
quent experiments. To further improve the peak symmetry of the com­
pounds, TEA was employed as mobile phase additive for silanol group
masking. Enhanced peak symmetry and separation efficiency (almost 20
% increase) were obtained by using an aqueous solution H3PO4 (25 mM)
adjusted to pH 3.0 with TEA in combination with acetonitrile. Under the
above conditions and despite the fact that the shape of the peaks was
acceptable, we were not able to achieve baseline separation of the
structurally similar CIP and NOR (Rs less than 1.5). The incorporation of
methanol in the mobile phase B (15 % v/v methanol) offered better
control of its eluotropic strength in the first isocratic step but the reso­
lution of CIP/NOR pair still remained less than 1.5.
The above-mentioned selectivity issue was addressed by studying the
effect of the column temperature [28]. The effect of the column tem­
perature was therefore investigated in the range of 25 – 50 ◦ C. Favor­
ably, increased column temperature at > 45 ◦ C resulted in enhanced
Microchemical Journal 172 (2022) 106906
resolution of CIP/NOR to > 2.2. The latter value has been selected for
subsequent studies.
,ν=
3.2. Optimization of the HLPME conditions
In this work HLPME was developed for the extraction of the selected
fluoroquinolones from human urine samples. To achieve the optimal
extraction, scheme several parameters were investigated. The assess­
ment of the type of extraction solvent and salt were initially performed
using one-factor-at-a-time (OFAT) approach following by a two-step
experimental design (screening and optimization) in order to conclude
to the optimal HLPME experimental conditions. In all related experi­
ments, an artificial spiked urine sample (concentration level of 1 μg
mL− 1) was used in order to avoid any potential interference from the
matrix on the performance of HLPME.
3.2.1. Type of the hydrophilic organic solvent and phase-forming agent
The mechanism of the proposed HLPME is based on two consecutive
steps. The first step includes the addition of a hydrophilic organic sol­
vent to the aqueous biological sample solution forming a homogeneous
phase. The second step, the phase separation and analyte extraction take
place after the addition of a phase-forming agent (salt). Selecting the
appropriate solvent is essential since it affects the overall extraction
(1) performance [21,29]. In aqueous biphasic systems the hydrophilic
organic solvent should have some general characteristics: i) high
extraction efficiency, ii) good chromatographic behaviour and iii) to
form a consistent clear layer after phase separation. Additionally, the
phase-forming agents should have negligible solubility in the hydro­
philic organic solvent and high solubility in water. In the case of a salt, it
should have high hydration enthalpy and its concentration should be
close to saturation [30].
Preliminary experiments were conducted to evaluate the appropri­
ateness of four hydrophilic organic solvents (logP indicated in the
parenthesis) namely acetone (5.1), acetonitrile (5.8), isopropanol (3.9)
and methanol (5.1) and six phase-forming agents (ZnSO4, MgSO4, NaCl,
NH4Cl, (NH4)2SO4 and Na2SO4). The HLPME procedure involved the
mixing of 500 μL of artificial urine (1:1 diluted) with equal volume of
each hydrophilic solvent followed by the addition of 200 μL of each salt
at concentration of 2 mol L-1.
In all trials, methanol could not be separated from the aqueous so­
lution. On the contrary, phase separation was promoted with the rest
organic solvents (acetonitrile, isopropanol and acetone) in combination
with sulfated salts such as MgSO4, Na2SO4 and ZnSO4. When using
MgSO4 and ZnSO4 as phase-forming agents the volume of the upper
organic layer was much higher than the remaining aqueous phase at the
end of the extraction. Analogous phenomena have also been reported in
the literature [31,32]. It should be noted that such a behavior may be
problematic in analysis of biological matrixes since it may lead to co-
extraction of polar potential interferences from the urine matrix. For
this reason, both MgSO4 and ZnSO4 salts were excluded. Quite similar
behavior was also observed when either isopropanol or acetone was
employed in combination with Na2SO4. Better results in terms of
extraction performance and sample cleanup was obtained by using
acetonitrile / Na2SO4 resulting in an upper organic layer with low water
content (450 μL ± 10%). Furthermore, acetonitrile offers better
compatibility with the acetonitrile-based HPLC mobile phase and was
therefore chosen throughout this study.
3.2.2. Screening of parameters using Plackett-Burman design (PBD)
A Plackett-Burman experimental design was applied in order to find
out the experimental parameters having significant effect on the
extraction efficiency of the analytes. On this basis, a two-level factorial
design was built to screen five plus two dummy factors including volume
of acetonitrile (X1), sample volume (X2), sample pH (X3), centrifugation
time (X4), salt concentration (X5). Eleven runs (including three exper­
iments at the center point) were conducted in a random manner to
4
E. Tsanaktsidou et al. Microchemical Journal 172 (2022) 106906

reduce the error. The investigated parameters, their levels and experi­
mental results are shown in Table 2S and Table 3S. The extraction re­
covery (ER %) was obtained as the response in all cases. Pareto ranking
plots (Fig. 1) were derived from multivariate regression analysis. The
bar length is proportional to the absolute value of the estimated stan­
dardized effects while the vertical red line judges the effects that are
statistically significant at the 95% confidence level. As it illustrated in
Fig. 1 the sample volume (Vs), the volume of acetonitrile (VACN) and the
Na2SO4 concentration (Csalt) were found statistically significant (p less
than 0.05) for all analytes except from PRU where the sample pH had
significant effect. Based on these findings and for the sake of simplicity
and uniformity, we decided to exclude the sample pH factor from the
optimization step leading to a smaller 3-factors Box-Behnken experi­
mental design. Additionally, the pH adjustment of the samples - prior to
their extraction – is a laborious procedure lowering sample throughput.
The centrifugation time was non-significant in the examined range (i.e.
1–5 min) and its value was set according to the sign on the Pareto charts.
Lastly, the three significant parameters (Vs, VACN and Csalt) were selected
for the optimization experiments.
“lack-of-fit” (LOF) was higher than 0.05 and therefore non-significant
associated to the pure error. Diagnostic plots (i.e normal probability
plot of residuals, plot of residuals against to the predicted values) are
shown in Fig. 2S (supplementary material). The data were monoto­
nously dispersed around the line; thus, there is a good correlation be­
tween the predicted and actual responses. A representative set of
response surface plots of MOX were illustrated in Fig. 2.
In order to find the optimum experimental conditions Derringer’s
desirability function (D) was applied scaled in the range of 0 to 1 as a
fully desirable response. The prediction profiles and the desirability
graphs are illustrated in Fig. 3. The optimum response for a single factor
at levels 0, 0.5 and 1 can be predicted by keeping the rest factors con­
stant at their center values of 0.5. The optimum values were found to be
200 μL for Vs, 500 μL for VACN and 2.5 mol L-1 for Csalt. Finally, to
confirm the optimum set of conditions, six repetitive extractions were
carried out. The differences between the obtained experimental values
with the predicted values were less than 8 %.
3.3. Method validation

3.2.3. Optimization of parameters using response surface methodology
The subsequent step includes the optimization of the three signifi­
cant parameters using response surface methodology through the Box-
Behnken design (BBD). BBD is an efficient tool widely used in the
optimization of microextraction techniques [33]. In our case a total of
20 experiments (5 runs in the center points) were performed keeping the
same experimental domain investigated at the screening test. The
factorial design points are tabulated in Table 4S and all the experiments
were randomized to minimize the systematic errors. By applying
multivariate regression analysis (the model is given in our previous
study [32]), a fitted second-order polynomial quadratic model was
constructed.
The experimental designs and statistical data analysis were accom­
plished using TIBCO Statistica software v. 13.3.0 (TIBCO software Inc.,
Palo Alto, CA, USA). The most important effects and interactions were
evaluated by ANOVA and the results are tabulated in Table 5S-8S. The
calculated R2 of the models were higher than 0.8517 indicating that the
predicted models can explain adequately the responses. The p-value of
The proposed method was validated using the accuracy profiles
based on tolerance intervals (section 2.4). The accuracy profiles are
represented in Fig. 4. The peak area ratio of each analyte to ISTD was
used in all cases. Due to the different responses of the analytes the
linearity was assessed in the range of 5 – 100 ng mL− 1 for CIP and NOR
and 50 – 1000 ng mL− 1 for MOX and PRU, respectively. The red and the
dashed blue lines symbolize the relative bias and the β-expectation
tolerance limits at 95% probability level, respectively. The β-expectation
tolerance intervals are estimated for each concentration level of the
validation standards. In the case of the dashed blue lines are comprised
within the black dotted lines the (predefined acceptance limits of 15%
for bioanalytical methods), the method can be considered as valid. All
validation results are presented in Table 1 and Table 9S-11S.
3.3.1. Selectivity and carryover
The selectivity of the developed method was assessed by analyzing a
pooled blank FQ-free (n = 6) and spiked human urine samples in order to
investigate the presence of potential interferences. No interfering peaks

Table 2
Overview of recent microextraction-based methods for the HPLC determination of FQs in biological samples.
Analytes Sample Sample Extraction procedure Detection LOD (ng Recovery Ref
type volume (μL) mL− 1) %

Ciprofloxacin, Ofloxacin, Norfloxacin, Enrofloxacin Serum/ 500 DMSPE1 HRMS 0.02 – 85 – 113 [12]
urine 0.03
2
Ciprofloxacin, Moxifloxacin Blood 1500 MIP-SPME MS/MS 0.13 – 65 – 83 [35]
0.20
Ciprofloxacin, ofloxacin, norfloxacin, levofloxacin Serum/ 2500 MIP-SPME UV 0.023 – 98–101 [36]
plasma 0.033
Ciprofloxacin, enrofloxacin, marbofloxacin, norfloxacin Urine Not PT-MIP-SPE3 UV 39 42–96 [37]
mentioned
4
Ofloxacin, ciprofloxacin, norfloxacin, enrofloxacin, fleroxacin Plasma/ 1000 Salt-induced LLME FLD 0.12 – 93 – 111 [17]
urine 0.66
Ciprofloxacin, norfloxacin, marbofloxacin, danofloxacin Urine 8.1 LPME on microchip UV 90 – 110 35 – 62 [14]
Flerofloxacin, ciprofloxacin Urine 8000 IL-LPME5 UV 3.12 – 82.3 – [16]
4.97 106.8
Danofloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, Urine 5000 SALLE FLD 0.14 – 58 74.7 – [18]
difloxacin, flumequine, enoxacin, oxolinic acid, moxifloxacin, 102.2
lomefloxacin, marbofloxacin, pipemidic acid, norfloxacin
Ofloxacin Urine 1000 Effervescence-assisted FLD 3.6 94 – 99 [19]
SALLE with switchable
solvent
Ciprofloxacin, norfloxacin, moxifloxacin, prulifloxacin Urine 200 HLPME FLD 3 – 11 97.2 – Present
107.7 method
1
DMSPE: dispersive micro solid phase extraction
2
MIP-SPME: molecularly imprinted polymer solid phase microextraction
3
PT-MIP-SPE: pipette-tip molecularly imprinted polymers solid phase extraction
4
LLME: liquid liquid microextraction
5
IL-LPME: ionic-liquid liquid phase microextraction

5
E. Tsanaktsidou et al.
Fig. 2. Response surface (3D) plots indicating the effects of A) VACN and Vsample, B) Csalt and Vsample and C) Csalt and VACN on the ER% of MOX.
from the biological matrix were observed in the elution region of the
analytes the ISTD considering the sufficient selectivity of the method.
Representative chromatograms of blank and spiked urine samples are
depicted in Fig. 5.
The potential carry-over effect of HPLC system was studied by sub­
sequently analyzing a blank and a spiked urine sample at the highest
calibration level. No influence on the signal was recorded indicating that
the “between-injection” washing procedures of the autosampler and the
column were satisfactory.
3.3.2. Linearity, LOD, LOQ
The linearity of the results expresses the ability of the analytical
method to provide results directly proportional to the concentrations. In
order to simplify the future calculations, simple linear unweighted
regression models were adopted for all analytes. With this calibration
model all concentrations gave results within the acceptance limits apart
from CIP and NOR (Fig. 5A and 5B). The high biases observed at the
lowest calibration level of 5 ng mL− 1 lead to β-expectation profiles above
the upper and lower acceptance limits (15%). Based on these results, the
lower limit of quantitation (LLOQ) level was set to 10 ng mL− 1 for CIP
and NOR considering that this level can provide accurate and repro­
ducible results. For the rest of compounds, the LLOQ level was fixed at
50 ng mL− 1 which is adequately low for bioanalytical applications. The
limit of detection (LOD) of the drugs were estimated as YLOD = intercept
+ 3 × residual SD obtained by ANOVA [34]. The back-calculation
provides XLOD for each series. The mean value of XLOD were 3, 3, 11
and 9 ng/mL for CIP, NOR, MOX and PRU, respectively.
6
Microchemical Journal 172 (2022) 106906
The linearity of the method was examined by fitting least squares
regression lines on the back-calculated concentrations of the validation
standards as a function of the introduced analyte concentration. Cali­
bration parameters (slope, intercept, correlation coefficient, etc) are
shown in Table 1 and Table 9S-11S. The linearity of the HLPME method
was also verified since the absolute β-expectation tolerance limits were
within the absolute acceptance limits (Fig. 3S supplementary material).
3.3.3. Trueness, accuracy and precision
Trueness is expressed as the mean bias (%) between the measure­
ments and the target concentrations. As reported in Table 1 and
Table 9S-11S the relative biases ranged between ─ 2.8 and 7.7 % for all
compounds indicating the good trueness of the method. Precision was
expressed as the RSD of repeatability (sr, %) and time-dependent inter­
mediate precision (sR, %) for each concentration level. In all cases, both
sr and sR values were lower than 6.6 %, demonstrating the adequate
precision of the proposed method. The upper and lower β-ETIs for each
level for FQs are completely included inside the acceptance limits of ±
15%. Therefore, the proposed analytical scheme can be considered as
accurate in the examined range.
3.3.4. Robustness using Monte-Carlo simulations and capability analysis
The robustness of the HLPME method was examined using Monte-
Carlo simulations and capability analysis. Based on this, 100,000 iter­
ations were performed using Monte-Carlo simulations experiments and
the simulated data were utilized to estimate the Cpk values. The
acceptance criteria of the ER of each analyte were fixed to ± 10 % of the
E. Tsanaktsidou et al. Microchemical Journal 172 (2022) 106906

Fig. 3. Profiles for predicted values and desirability function (red colored lines designate the optimum value).

Fig. 4. Accuracy profiles for the FQs determination in human urine using a linear unweighted regression model for A) CIP, B) NOR, C) MOX and D) PRU. The red
plain, blue dashed and blank dotted lines correspond to the relative error (%), the accuracy profile and the acceptance limits λ (±15%), respectively.

7
E. Tsanaktsidou et al. Microchemical Journal 172 (2022) 106906

1
Fig. 5. Representative chromatogram of the analysis of blank (black line) and spiked urine sample with ISTD (fuchsia line) and FQ standard mixture at 100 ng mL−
after HLPME.

predicted value obtained from the optimization step (section 3.2.3). The
set of simulations were conducted considering the mean value of 200,
500 and 2.5 mol L-1 for the Vs, VACN, Csalt with SD of, 5, 5 and 0.1,
respectively. Fig. 6 illustrates the histograms from the capability anal­
ysis of the studied response for each drug. The results from the capability
analysis led to CpK values of higher than 2.286 which are much higher
than the acceptance limit of 1.33.
3.3.5. Sample stability
The stability of FQ in unprocessed urine samples was assessed at
ambient temperature, 4 ◦ C and –18 ◦ C up to 24 h. The experiments shown
that FQ were stable up to 24 h when the urine samples were stored at 4 ◦ C
and –18 ◦ C (the recoveries were > 90 % for each analyte). However, the
sample storage at ambient temperature resulted in significant degrada­
tion of all compounds up to 8 h (e.g PRU recovery was 18 %).

Fig. 6. Probabilistic distribution of the extraction recoveries of the A) CIP, B) NOR, C) PRU and D) MOX during Monte-Carlo simulation experiments.

8
E. Tsanaktsidou et al.
3.4. Comparison with other reported methods
The proposed HLPME method was compared with other previously
reported microextraction-based methods such as DMSPE [12], MIP-
SPME [35,36], PT-MIP-SPE [37], LPME on microchip [14], salt-
induced LLME [17], IL-LPME [16] and effervescence-assisted liquid
phase microextraction with switchable solvent [19]. An overview of the
main features of these methods is presented in Table 2. In terms of
analytical figures of merit all the tabulated methods seem to be capable
of determining the selected quinolones in biological material, with the
sensitivity being closely related to the detection system (e.g. MS vs UV)
and the preconcentration potentials of the sample preparation step. On
the other hand, practical applicability of each method is limited by
several factors such as for example the availability of MIP materials
[35–37] or the preparation of microchip devices [14]. Our proposed
method can be readily applied to any laboratory, with common reagents
and without the need of the fabrication of dedicated configurations for
the collection of the extractant [17] or complicated flow-based setups
[19]. It also utilizes a comparatively small volume of sample (200 μL)
that is perfectly adoptable to alternative matrices such as plasma or
serum.
4. Conclusions
In this study, a simple and fast microextraction method based on the
usage of hydrophilic media in combination with salt was developed to
extract fluoroquinolones from human urine matrixes prior to their
determination by HPLC-FLD. A small amount of 500 μL of acetonitrile
was required for the whole procedure instead of highly toxic haloge­
nated organic solvents which is in accordance with the principles of
green analytical chemistry. The extraction conditions were optimized by
experimental design based on PBD and BBD in order to have a sensitive
method. The validation of the method was performed using accuracy
profiles approach by testing several criteria (trueness, precision, accu­
racy, linearity, limits of detection and quantitation). The relative bias
ranged between ─ 2.8 to 7.7 % for all FQs, while low LODs (3 – 11 ng
mL− 1) were achieved in urine samples. The method proved to be robust,
while the performance and validation data are satisfactory for routine
applications in pharmacokinetic studies.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.microc.2021.106906.
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