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Michał Pieckowski Piotr Kowalski iD Ilona Olędzka Natalia Miękus-Purwin Alina Plenis Anna Roszkowska Tomasz Bączek
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gdańsk, Poland
Received August 17, 2021 Revised September 17, 2021 Accepted September 30, 2021
Research Article
Simultaneous determination of mitotane, its metabolite, and five steroid hormones in urine samples by capillary electrophoresis using ß-CD2SDS1 complexes as hydrophobic compounds solubilizers
Mitotane is a cytotoxic drug used in the treatment of inoperable adrenocortical carcinoma, it inhibits steroidogenesis as well, and therefore monitoring the level of steroid hormones in patients treated with mitotane is a crucial point of therapy. Hence, we have developed a simple, fast, and efficient electrophoretic method combined with reverse polarity sweeping as online preconcentration technique and dispersive liquid-liquid microextraction for the simultaneous determination of mitotane, its main metabolite DDA, and five steroid hor- mones (progesterone, testosterone, epitestosterone, cortisol, and corticosterone) in urine samples. In addition, a new sample matrix consisting of ß-CD2SDS1 complexes for a high hydrophobic compounds solubilization was developed. Approach based on the application of ß-cyclodextrin and SDS complex of a ratio 2:1 allowed for hydrodynamic injection into the capillary of a solution containing both mitotane and other analytes. The detection limits of the analytes for the reverse polarity sweeping-dispersive liquid-liquid microextraction method were found to be in the range of 1.5-3 ng/ml, which were approximately 1000 times lower than in the conventional hydrodynamic injection (5 s, 0.5 psi) without any preconcentration procedure. All analytes were completely resolved in less than 13 min by uncoated silica capillary with an inner diameter of 75 µm (ID) x 60 cm. Electrophoretic separation was performed in reverse polarity with a voltage of -25 kV with a background electrolyte (BGE) consisting of 100 mM SDS, 25% ACN, 25 mM phosphate buffer (pH 2.5), and 7 mM ß-cyclodextrin.
Keywords:
Adrenocortical carcinoma / -Cyclodextrin / Dispersive liquid-liquid microextrac- tion / Hydrophobic compounds solubilization / Online preconcentration tech- niques / Sweeping DOI 10.1002/elps.202100250
1 Introduction
Majority of adrenal cortex carcinomas (ACC) are charac- terized by excess hormone secretion (60% of all cases) [1]. Among tumors with hormonal activity, the most frequent is the excessive secretion of cortisol (50-80%), however there are cases of excessive secretion of androgens (40-60%) and estrogens (6-10%). Control of the secretion of both cortisol and testosterone should be implemented in every patient with ACC, moreover 24 h urinary free cortisol is recommended hormonal evaluation in patients with suspected ACC. Mea- surement of the concentrations of other steroid hormones
Correspondence: Professor Piotr Kowalski, Department of Phar- maceutical Chemistry, Medical University of Gdańsk, Hallera 107, 80-416 Gdańsk, Poland
E-mail: piotr.kowalski@gumed.edu.pl
Abbreviations: ACC, adrenocortical carcinoma; DLLME, dis- persive liquid-liquid microextraction; IS, internal standard; RP, reverse polarity; SM, sample matrix; B-CD, B-cyclodextrin
is optional, however, their elevated values may be considered as markers for the use of additional hormone inhibitors or for tumor recurrence [2,3]. Mitotane (1-(2-chlorophenyl)- 1-(4-chlorophenyl)-2,2-dichloroethane, o, p”-DDD) is a chlorinated bicyclic aromatic compound used in the treat- ment of inoperable adrenal cortex cancer and to reduce the risk of recurrence and metastasis after tumor resection. The mechanism of action of mitotane is based on both anti- neoplastic and anti-hormonal activity; however, the cytotoxic effect of mitotane has not been entirely known [1]. Mitotane influences steroidogenesis as well as the metabolism of adrenal cortex hormones by inhibiting such enzymes as 20,22-desmolase, 11ß-hydroxylase, or 3-hydroxysteroid dehydrogenase [4]. Such an extensive effect on the endocrine system requires the monitoring of steroid levels in patients treated with mitotane [5]. Several methods for the determi- nation of mitotane have already been developed using HPLC
Color online: See article online to view Fig. 1 in color.
| Method | Analyte | LOD | LOQ | Matrix | Reference |
|---|---|---|---|---|---|
| HPLC-UV | Mitotane, p,p'-DDA, DDE, p,p'-DDD(IS) | – | 0.2-0.5 µg/mL | Plasma | [6] |
| HPLC-UV | DDT, mitotane, DDE, DDA, dialdrin (IS) | 0.3 µg/mL | 0.5 µg/mL | Plasma, red cells | [7] |
| HPLC-LLE-UV | Mitotane, DDE, aldrin(IS) | 36-102 µg/mL | 108-310 µg/mL | Plasma | [8] |
| HPLC-SPE-DAD | Mitotane, DDE, DDA | 0.050-0.132 µg/mL | 0.145-0.399 µg/mL | Plasma, red cells, urine | [9] |
| HPLC-SPE-UV | Mitotane, DDE, DDA, DDT (IS) | 0.2 µg/mL | 0.4 µg/mL | Plasma | [10] |
| GC-EI-MS | Mitotane | - | 0.25 µg/mL | Plasma | [11] |
| CE-CD-MEKC- | Mitotane, DDA, progesterone | 1-3 ng/ml | 5-10 ng/ml | Urine | This study |
| DLLME | testosterone, epitestosterone, corticosterone, cortisol, dexamethasone (IS) |
LLE: liquid-liquid extraction; DDE: 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene; DDT:
1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane; IS: internal standard.
[6,7], combined with liquid-liquid extraction [8], and SPE [9,10] (see Table 1). Ando et al. developed the determination of mitotane in blood plasma samples by GC in combination with electron ionization-MS [11]. Thus far, electrophoretic method has not been developed for o, p”-DDD and its metabolites; moreover, the simultaneous determination of mitotane and steroid hormones has not been obtained as well. Likewise, there are insufficient data in the literature on the levels of DDD and DDA in patient urine samples. Mornar et al. [9] conducted studies on urine samples of patient treated with 1.5 g of mitotane (500 mg dose taken every 8 h). The detected concentration of mitotane was 710 ng/ml; however, DDA was not present in the urine sample as explained by the lack of mitotane biotransformation. Chen et al. [12] stud- ied DDA concentrations in urine samples in a population exposed to the insecticide 1,1,1-trichloro-2-(2-chlorophenyl)- 2-(4-chlorophenyl)ethane, a compound of the same group as mitotane, a bicyclic aromatic chlorinated hydrocarbon, using GC. The concentration of DDA, which is a metabolite of 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane, ranged from 11 to 92 ng/ml in urine samples. For those reasons, this study developed a fast and sensitive dispersive liquid-liquid microextraction (DLLME) of mitotane, its acetic acid metabolite 1,1-(o, p”-dichlorophenyl) (o, p”-DDA), and five steroid hormones (progesterone, testosterone, epitestos- terone, cortisol, and corticosterone) from urine samples with electrophoretic separation using UV detection and sweeping as an online preconcentration technique. Moreover, the use of the spontaneous formation of B-CD2SDS1 complexes in aqueous solutions and their influence on the solubility of hydrophobic analytes was investigated. Therefore, in this work practically insoluble mitotane was selected as a model of highly hydrophobic drug to study the possibility of online preconcentration in the capillary, in the presence of B-CD2SDS1 complexes in the sample matrix. Furthermore, the influence of standard electrophoretic parameters such as sample volume, applied voltage, composition and pH of the separation buffer, and the concentration of the organic modifier were optimized. The impact of SDS concentra-
tion on the potential of the sweeping technique was also examined.
2 Materials and Methods
2.1 Chemicals and reagents
o,p’-DDD (M), DDA, steroids (progesterone [P], testosterone [T], epitestosterone [E], corticosterone [C1], cortisol [C2]), in- ternal standard ([IS], dexamethasone), SDS, urea, uric acid, sodium phosphate monobasic dihydrate (NaH2PO4 . 2H2O), potassium dihydrogen phosphate anhydrous (KH2PO4), and HPLC-grade ACN were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphoric acid (H3 PO4), sodium chloride (NaCl), potassium chloride (KCI), and ß-cyclodextrin (B-CD) were purchased from Merck (Darmstadt, Germany). Highly pure water (distilled and deionized) was obtained from the Milli-Q equipment (Millipore, Bedford, MA, USA).
2.2 Urine samples
In our study, the synthetic urine solution was used for the estimation of validation parameters, dilution integrity, and carry-over; on the other hand, urine collected from healthy volunteers was used for the development of the extraction conditions, as well as selectivity and stability. Synthetic urine solution was prepared by dissolving 3.034 g of urea, 0.042 g of uric acid, 2.0 of NaCl, 0.050 g of KCl, 0.285 g of NaH2PO4.2H2O, and 0.050 g of KH2PO4 (anhydrous) in 250 ml of deionized (18.2 MS2 cm) water, and finally adjusted to pH 6.5 by 0.1 M NaOH solution.
2.3 Analytes standard solutions
Individual stock solutions of analytes at a concentration of 2 mg/mL were prepared in pure ACN and stored at -20℃ in
dark containers to avoid the decomposition. Standard and working solutions (5, 0.25, 0.025 µg/mL) were freshly pre- pared at each working session from the stock solutions by proper dilution with pure ACN. All working solutions were stored at 4℃ in dark containers for 48 h.
2.4 CE instrument
The electrophoretic separations were conducted on a PACE/MDQ capillary electrophoresis system (Beckman In- struments, Fullerton, CA, USA) equipped with a DAD op- erating at 200 nm, 214, or 248 nm and controlled with 32 Karat 8.0 software. Uncoated fused-silica capillaries (Polymi- cro Technology) with an id of 75 um and 60 cm total length (50 cm length to the detector) were used for method devel- opment. The capillary temperature was controlled at 25.0 (± 0.1)℃ during all experiments. To ensure good reproducibil- ity of analytes, the new capillary was conditioned with 1 M NaOH (10 min), 0.1 M HCl (10 min), and deionized water (10 min), and with the separation buffer for 10 min prior to use. Between each run, the capillary was washed with 0.1 M NaOH (2 min), deionized water (2 min), and finally with the separation buffer for 2 min. The pH values of the separation buffers and other solutions were measured using a pH meter (Beckman Coulter, CA, USA).
2.5 Extraction procedure
In the reverse polarity sweeping (RP-sweeping)-DLLME method, 5 mL of urine samples filtered by Amicon Ultra-15 Centrifugal Filter with 10.000 NWML membrane were spiked with selected steroid hormones, mitotane, DDA, and an in- ternal standard from working solutions with concentrations of 250 ng/ml and 25 ng/ml. Then, 100 µL of 0.1M HCI was added to the samples and vortexed, next 1100 µL of the dispersion mixture, composed of the mixture of ACN and dichloromethane in the ratio 9:2 (v/v), was rapidly injected to the samples. The cloudy solution was shaken vigorously and centrifuged for 8 min at 4350 rpm. Dichloromethane phase was collected with a Hamilton syringe, transferred to Eppen- dorf tubes, and evaporated in a vacuum centrifuge to obtain a dry precipitate. The precipitates were dissolved in 10 µL of ACN, and subsequently in the 90 µL sample buffer system, thus the final concentration was 3.5 mM SDS, 7 mM B-CD, and 60 mM H3 PO4. The prepared samples were centrifuged for 2 min at 14 800 rpm and electrophoretically separated by sweeping procedure.
3 Results and Discussion
3.1 Sample matrix composition
Due to the significant lipophilicity of analytes, in particular mitotane (logP = 6.11) [13], the development of sample
+
SDS monomer
ß-cyclodextrin
ß-CD2SDS1 complex
matrix composition for the electrophoretic separation was a crucial point of analysis procedure optimization. We found ACN to be the optimal solvent for all analytes, however concentrations major than 30% (v/v) disrupted the current during separation. On the other hand, the presence of water in the sample matrix (SM) caused the precipitation of mi- totane. For this reason, it was necessary to use solubilizers such as SDS or cyclodextrins, although due to the limitations of the sweeping method (the matrix must contain signifi- cantly less SDS than the separation buffer), it was not possible to use SDS at the concentration required to dissolve mitotane (concentration exceeding 20 mM). Only the addition of B- cyclodextrins made it possible to reduce the concentration of SDS to 3.5 mM and mechanism of this phenomenon consists of the formation of inner complexes between cyclodextrins particles and SDS monomers, which are stabilized by Van der Waals interactions [14]. - CD molecules spontaneously form an inclusion complex with SDS in a molar ratio of 2:1 in aqueous solutions, where one alkyl chain of SDS is embedded in two cavities of B-CD, forming a -CD2SDS1 complex [15] (Fig. 1). Lin et al. confirmed that increasing concentrations of cyclodextrins cause an increase in the CMC of SDS [16], which in the molecular mechanism results from the formation of B-CD2SDS1 complexes and their inability to micellize. Therefore, destacking phenomenon does not occur due to the absence of micelles in the sample matrix and sweeping preconcentration is not disrupted. Second, the pH value and conductivity of the sample significantly influenced the sharpness of the peaks, therefore phosphoric acid concen- trations in the range of 5-100 mM were examined. Due to the presence of ionizable chemical groups among the steroids and DDA, too low concentrations of H3PO4 did not ensure the appropriate conditions to obtain the signal amplification effect for all analytes. Likewise, higher acid concentrations increased the conductivity, resulting in sharper peaks, nonetheless above 70 mM destabilized the electric field, therefore concentration of 60 mM H3PO4 in the SM ensured acceptable peak sharpness and current stability. Conclusively, the optimized sample matrix consisted of 10% ACN, 3.5 mM SDS, 7 mM B-CD, and 60 mM H3 PO4 with a conductivity ratio SM/BGE = 1.2.
Absorbance (AU)
0.12
M+DDA+P+E+T+C1
UV = 214 nm
0.11
C2
0.10
A
0.09
EI
T
C1
0.08
IS
C2
0.07
M+DDA+P
[5 mAU
B
0.06
E
T
0.05
C1
IS
C2
0.04
DDA+P
C
M
0.03
P
E
IT
0.02
C1
IS
C2
0.01
DDA
D
M
0.00
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
Time (minutes)
14.0
3.2 Buffer composition and sweeping mechanism
The RP-sweeping mechanism is based on the suppression of electroosmotic flow by the acid (25 mM H3PO4, pH 2.5), whereby the analytes are “swept” and stacked by SDS mi- celles that migrate toward the anodic end of the capillary. The RP-sweeping method, despite significant signal ampli- fication in the case of hydrophobic compounds, due to their high affinity for hydrophobic micelle cores, is characterized by relatively low resolution. Therefore, it has proved neces- sary to use organic modifiers for the complete separation of the analytes. As shown in Fig. 2, the background electrolyte consisting of 25 mM H3PO4, 100 mM SDS maintained an exceptionally low resolution, and the addition of ACN signif- icantly influenced the migration time. However, at its 25% (v/v) content in the running buffer all analytes were separated except for DDA and progesterone, and likewise higher con- centrations of ACN disrupted current stability. Solutions con- taining over 30% ACN cause substantially complete precipi- tation of the ß-CD, and as a result of interruption of current flow.
On the other hand, 7 mM B-CD in combination with 25% ACN (Fig. 2D) provided high resolution due to the high affinity of the analytes to the hydrophobic B-CD core; as a result, the analytes were separated according to micellar electrokinetic chromatography technique modified with cy- clodextrins (CD-MEKC). In addition, we investigated the ef- fect of the concentration of SDS in the separation buffer on the sweeping potential. Figure 3 shows that increasing con- centrations of SDS affect peak sharpness, signal intensity, and analyte migration time. SDS concentrations exceeding 100 mM (Fig. 3E), despite the increase in signal, disturbed the baseline stability, therefore 100 mM SDS (Fig. 3D) was the
optimal concentration in terms of migration time and peak height for all analytes.
3.3 Extraction and disperser solvents
Extraction conditions were significantly related to the vari- ety of lipophilicity of the analytes and the presence of ion- izable chemical groups. The acidic nature of the DDA as well as the carboxyl and hydroxyl groups in steroids required acidification of urine samples with HCl to neutralize the ionic charge of the analytes and improve the extraction ef- ficiency to lipophilic solvents. Moreover, a study of the type of extractants and disperser solvents was examined. First, the extractants efficiency (dichloromethane and chloroform) from aqueous solutions spiked with the mixture of analytes (250 ng/ml) was investigated. After extraction with chloro- form, the peak signals were significantly low, while extrac- tion with dichloromethane provided a higher signal for all analytes, whereby dichloromethane was chosen as the ex- tractant. Second, four mixtures of dispersing solvents with dichloromethane were studied: acetone, ACN, methanol, and isopropanol. ACN has the best extraction performance for all analytes; hence it was selected as the disperser solvent. Furthermore, the volume of the extractant and disperser sol- vent on the extraction efficiency was investigated in the range of 100-300 and 500-1000 µ L-1, respectively. Volumes higher than 200 µL reduced the effectiveness of all analytes, except for cortisol, which showed an increase in peak height. Like- wise, ACN volumes below 900 µ L did not provide a sufficient dispersion of dichloromethane, while 1000 µL caused lower signal intensity, therefore the extraction mixture consisted of
Absorbance (AU)
0.14
P
E
T
UV = 214 nm
DDA
C1
0.12
A
M
IS
P
E
T
C1
IS
C2
0.10
B
M
DDA
P
E
T
C1
LIS
C2
0.08
C
DDA
M
P
E
T
C1
0.06
IS
C2
DDA
D
M
10 mAU
0.04
P
E
T
C1
IS
C2
0.02
DDA
E
M
0.0
5.0
7.0
9.0
11.0
13.0
15.0
17.0
19.0
Time (minutes)
ACN and dichloromethane in a 9:2 ratio (v/v) and a volume of 1100 µ L.
3.4 Validation study
The optimized RP-sweeping-CD-MEKC-DLLME method has been validated according to the European Medicines Agency guidelines for the validation of bioanalytical methods [17] by measuring peak heights at the wavelength of maximum ab- sorbance: 248 nm for steroids and 200 nm for mitotane and its metabolite.
3.4.1 Selectivity
To confirm the absence of interfering substances about the migration time of analytes, the blank urine and synthetic urine samples (n = 6) were analyzed and compared to matri- ces spiked with analytes and IS (25 and 100 ng/ml, respec- tively). As shown in Fig. 4, there is no significant urine matrix interference at migration time of analytes, hence the sample clean-up procedure was selective. Note that selectivity valida- tion was tested with 5 mL randomized urine samples, while the concentration of steroids is relatively lower than the 24 h urine aliquot, which is preferred method to control mitotane steroidogenesis inhibition.
3.4.2 Linearity, LOD, and LOQ
The LOD and LOQ were calculated via the value of the SD from the maximum sensitivity provided by the elec-
trophoretic system. The LOD and LOQ were estimated by multiplying the SD of the blanks by a factor of 3 and 10, respectively. Subsequently the resulting LOD and LOQ were validated separately by analyzing six samples from different standards (n = 6) (Table 2). The linearity of the method was determined by adding analytes to synthetic urine matrix in the range of 10-750 ng/ml for cortisol, 5-500 ng/ml for the rest of the analytes, and 100 ng/mL for internal standard in six replications (n = 6) of seven concentration. The relative peak heights were selected as analytical signals for all the an- alytes. The calibration curves were obtained by least squares linear regression analysis. Responses to mitotane, DDA, and steroids were linear in the concentration range studied (Table 2), which was confirmed by a satisfactory coefficient of determination (R2 ≥ 0.9988).
3.4.3 Accuracy and precision
The within-run accuracy and precision were calculated at LLOQ, LQC, MQC, and HQC levels for the five replicates, each of the same analytical run. For the equivalent concen- trations, accuracy and precision between runs over three dif- ferent analytical cycles in two different days were calculated. Based on the data presented in Table 3, it was confirmed that the values were within the acceptable limits according to rec- ommended guidelines.
3.4.4 Dilution integrity and carry-over
For dilution integrity, blank synthetic urine samples were spiked with two times higher than the ULOQ (upper limit of
Absorbance (mAU)
40.0
UV = 248 nm
35.0
30.0
P
E
T
25.0
C1
IS
C2
20.0
A
1
1
1
1
IS
1
15.0
P
E
T
C1
C2
B
10.0
IS
5.0
C
1
1
1
+
4
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
Time (minutes)
Absorbance (mAU)
40.0
UV = 200 nm
35.0
DDA
30.0
M
IS
25.0
A
20.0
IS
15.0
B
10.0
IS
C
1
5.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
Time (minutes)
quantification) concentration (1500 ng/ml and 1000 ng/ml for cortisone and rest of the analytes, respectively) and was diluted 5 times and 10 times with blank synthetic urine (n = 6) to obtain a concentration of 300 ng/mL and 150 ng/ml for cortisone, as well as 200 ng/ml and 100 ng/ml for other analytes. Diluted samples were assayed alongside the deter- mined calibration curve. The results of dilution integrity in- vestigation were within acceptable limits. The accuracy (%) and precision (%CV) were found to be in 93.0-106.4 and 5.5- 8.1 range, respectively, which is within the limit of the stan- dard accepted by the guidelines, that is, ±15%. Carry-over ef- fects were assessed by injecting to the capillary three blank synthetic urine samples, one before, and two after ULOQ cal- ibration standard (750 ng/mL for cortisone and 500 ng/ml
for other analytes) injection, consequently no carry-over ef- fects were observed.
3.4.5 Stability
During the validation process, the stability of the analytes in stock solutions and urine samples was tested in triplicate (n = 3) LQC and HQC. All stability determinations were analyzed against a freshly prepared calibration curve to obtain sample concentrations. The samples have been sub- jected in short-term stability at room temperature for 6 h; postpreparative stability for 24 h at room temperature; after three cycles of freeze (-70 ℃) and thaw; long-term stability
| Analyte | Linear range(ng/mL) | Equation | R2 | LOD(ng/ml) | LOQ(ng/ml) |
|---|---|---|---|---|---|
| Mitotane | 5-500 | y = (0.0337 ± 0.0005)x- (0.0821 ± 0.1045) | 0.9990 | 1.5 | 5.0 |
| DDA | 5-500 | y=(0.0479 ± 0.0004)x-(0.1186 ± 0.0892) | 0.9996 | 1.5 | 5.0 |
| Progesterone | 5-500 | y = (0.0234 ± 0.0004)x+ (0.0177 ± 0.0785) | 0.9988 | 1.5 | 5.0 |
| Epitestosterone | 5-500 | y = (0.0326 ± 0.0003)x+ (0.0046 ± 0.0716 | 0.9995 | 1.5 | 5.0 |
| Testosterone | 5-500 | y = (0.0275 ±0.0003)x+ (0.0069 ± 0.0619) | 0.9995 | 1.5 | 5.0 |
| Corticosterone | 5-500 | y=(0.0248 ± 0.0002)×+ (0.0021 ± 0.0483) | 0.9996 | 1.5 | 5.0 |
| Cortisol | 10-750 | y=(0.0118 ±0.0002)x+ (0.0215 ± 0.0582) | 0.9990 | 3 | 10.0 |
| Analyte | Nominalconcentration | Within-run (n = 5) | Between-run (n= 6) | ||||
|---|---|---|---|---|---|---|---|
| Observed- concentration (ng/ml) (mean ± SD) | Accuracy (%) | Precision (CV%) | Observed- concentration (ng/ml) (mean ± SD) | Accuracy (%) | Precision (CV%) | ||
| Mitotane | LLOQ | 4.78 ± 0.54 | 93.6 | 11.6 | 4.60 ± 0.45 | 92.1 | 9.9 |
| LQC | 15.66 ± 1.25 | 104.4 | 8.0 | 14.00 ± 1.19 | 93.3 | 8.5 | |
| MQC | 197.15 ± 13.87 | 98.6 | 7.0 | 213.31 ± 14.31 | 106.7 | 6.7 | |
| HQC | 461.89 ± 30.97 | 102.6 | 6.7 | 475.92 ± 21.23 | 105.8 | 4.5 | |
| DDA | LLOQ | 5.34 ± 0.71 | 106.8 | 13.4 | 4.84 ± 0.63 | 96.8 | 12.9 |
| LQC | 16.03 ± 1.00 | 106.9 | 6.3 | 15.77 ± 1.05 | 105.1 | 6.7 | |
| MQC | 202.38 ± 9.69 | 101.2 | 4.8 | 211.66 ± 14.80 | 105.8 | 7.0 | |
| HQC | 440.21 ± 23.19 | 97.8 | 5.3 | 444.30 ± 18.31 | 98.7 | 4.1 | |
| Progesterone | LLOQ | 4.66 ± 0.53 | 93.1 | 11.3 | 5.48 ± 0.61 | 109.5 | 11.1 |
| LQC | 14.29 ± 1.41 | 95.2 | 9.9 | 16.34 ± 1.96 | 108.9 | 10.1 | |
| MQC | 206.17 ± 11.45 | 103.1 | 5.6 | 188.13 ± 11.54 | 94.1 | 6.2 | |
| HQC | 474.97 ± 24.89 | 105.6 | 5.2 | 456.37 ± 24.54 | 101.4 | 5.4 | |
| Epitestosterone | LLOQ | 4.61 ± 0.68 | 92.2 | 14.7 | 5.38 ± 0.48 | 107.5 | 9.0 |
| LOC | 15.85 ± 1.36 | 105.7 | 8.6 | 15.15 ± 0.77 | 101.0 | 5.1 | |
| MQC | 206.87 ± 14.30 | 103.4 | 6.9 | 209.00 ± 9.56 | 104.5 | 4.6 | |
| HQC | 461.02 ± 15.18 | 102.5 | 3.3 | 445.00 ± 13.55 | 98.9 | 3.1 | |
| Testosterone | LLOQ | 5.40 ± 0.65 | 108.1 | 12.1 | 4.72 ± 0.56 | 94.4 | 11.8 |
| LQC | 14.66 ± 1.13 | 97.7 | 7.7 | 14.42 ± 1.47 | 96.1 | 10.2 | |
| MQC | 211.17 ± 15.40 | 105.6 | 7.3 | 210.90 ± 17.88 | 105.5 | 8.5 | |
| HQC | 462.95 ± 22.75 | 102.9 | 4.9 | 453.45 ± 18.81 | 100.8 | 4.2 | |
| Corticosterone | LLOQ | 4.59 ± 0.60 | 91.9 | 13.1 | 4.75 ±0.40 | 95.1 | 8.5 |
| LOC | 14.27 ± 1.41 | 95.1 | 9.9 | 14.30 ± 1.23 | 95.3 | 8.6 | |
| MQC | 209.19 ± 6.84 | 104.6 | 3.3 | 199.78 ± 16.98 | 99.9 | 8.5 | |
| HQC | 461.61 ± 26.75 | 102.6 | 5.8 | 424.38 ± 20.17 | 94.3 | 4.8 | |
| Cortisol | LLOQ | 10.55 ± 1.20 | 105.5 | 11.4 | 9.19 ± 0.85 | 92.0 | 9.2 |
| LOC | 31.23 ± 3.05 | 104.1 | 9.8 | 32.36 ± 2.59 | 107.9 | 8.0 | |
| MQC | 289.19 ± 25.69 | 96.4 | 8.9 | 313.43 ± 19.25 | 104.5 | 6.1 | |
| HQC | 616.82 ± 24.67 | 94.90 | 4.0 | 665.41 ± 28.44 | 102.4 | 4.3 | |
CV: coefficient of variation; LLOQ: lower limit of quantification (10.0 ng/ml for cortisol, 5.0 ng/ml for other analytes); LQC: lower quality control quantification (30.0 ng/ml for cortisol, 15.0 ng/ml for other analytes); MQC: medium quality control quantification (300.0 ng/ml for cortisol, 200.0 ng/ml for other analytes); HQC: high-quality control quantification (650.0 ng/ml for cortisol, 450.0 ng/ml for other analytes).
at -70℃; stock solutions of analytes stored for 120 days at -20℃; and IS solution stored for 30 days at 4-8℃. The stability study of mitotane and steroids in stock solutions and urine samples showed reliable stability as the mean of the
test samples results was within the acceptance criteria ±15% of the initial control values. These results indicate that the analytes can be handled under normal laboratory conditions without significant losses in routine analysis.
4 Concluding remarks
Mitotane is a highly lipophilic drug that has been deter- mined in biological samples by HPLC and GC methods. However, the aim of the study was to develop the first electrophoretic method for the determination of mitotane and its metabolite o, p’-DDA in urine samples. Due to the inhibition of steroidogenesis by mitotane, beside of the main drug and its metabolite five additional steroids were determined simultaneously. Likewise, for the first time the use of ß-CD2SDS1 complexes in CE to dissolve strongly hy- drophobic analytes in a (water-based) sample matrix has been described. Therefore, B-CD2SDS1 solubilization allowed the use of low concentrations of SDS in the sample that did not cause the phenomenon of destacking of the analyte peaks. Moreover, dispersive liquid-liquid microextraction provided an off-line signal amplification, low consumption of organic solvents, and the technique was significantly less expensive than liquid-liquid extraction or SPE (liquid-liquid extraction reagents or SPE columns) used in the determination of mitotane in biological samples. The developed method is simple, sensitive, and economical, with high throughput as well as environmental friendly. In addition, we conducted a full validation confirming that the precision and accuracy of the method meets the criteria of the guidelines, and there- fore can be used in clinical practice and routine analyses. Obtained parameters such as LOQ = 5 ng/ml and LOD = 1.5 ng/mL proved to be competitive with standard methods based on HPLC in combination with UV detection (Table 1).
The authors have declared no conflict of interest.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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