233 Current Pharmaceutical Analysis, 2018, 14, 233-238
RESEARCH ARTICLE ISSN: 1573-4129 eISSN: 1875-676X
A Rapid, Direct and Validated HPLC- Fluorescence Method for the Quantification of Abiraterone and Abiraterone Acetate in Urine and Serum Samples from Patients with Castration- Resistant Prostate Cancer
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BENTHAM SCIENCE
Juana Rodriguez1,*, Gregorio Castañeda1, Isabel Lizcano1 and Jose C. Villa2 1 2
Department of Analytical Chemistry and Food Technology, University of Castilla La Mancha. Ciudad Real, Spain; Department of Clinical Oncology, General University Hospital of Ciudad Real, Ciudad Real, Spain Abstract: Background: An accurate HPLC-fluorescence method for the simultaneous determination of abiraterone acetate (prodrug) and abiraterone (drug) employed in the treatment of prostate cancer, in urine and serum samples of men treated with this prodrug has been proposed.
ARTICLE HISTORY Received: November 10, 2016 Revised: December 16, 2016 Accepted: January 25, 2017
Methods: The developed HPLC-FLD procedure permits the quantification of ABR and AA minimizing laborious and complicated sample preparation procedures. The selectivity of the fluorescence detector avoids the presence of endogenous and exogenous interfering compounds and has enough sensibility for this determination.
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DOI: 10.2174/1573412913666170213152002
Results: The method was applied for the analysis of human urines samples of five men and serum samples of two men under treatment of prostate cancer. The excretion urinary in a patient was done during 24 hours. The maximum abiraterone concentration for this patient was 27 ng/mL.
Conclusion: This method shows the advantage of being suitable for the analysis of urine and serum samples of patients with prostate cancer who received daily doses of Zytiga.
Keywords: Abiraterone acetate, abiraterone, HPLC, fluorescence detection, biological fluids, prostate cancer. 1. INTRODUCTION
Prostate cancer is the second leading cause of cancerrelated deaths in developed countries which affect 16% of men [1]. Androgen deprivation therapy is the standard treatment for advanced stages of the disease and involves surgical or medical intervention. However, all patients eventually develop metastatic castration resistant prostate cancer (mCRPC) [2]. Abiraterone (ABR) (Fig. 1) is a highly potent, selective, irreversible inhibitor of 17- hydroxylase/C17, 20 lyase (CYP17) [3]. ABR catalyzes critical reactions in the synthesis of androgens in testicular, adrenal, and prostatic tumor tissues [4]. A prodrug, abiraterone acetate (AA) was marketed under the trade name Zytiga, approved by U.S. Food and Drug Administration (FDA) in April 2011 for the treatment of patients with mCRPC [5]. The recommended dose of AA is 1,000 mg (once daily) [6]. After oral administration, AA is extensively metabolized and ester hydrolysis was the primary biotransformation pathway, followed by further hydroxylation, sulfation and Noxidation, [7]. AA should be taken in fasting state since its bioavailability may increase up to 10-fold when it is administered after a high-fat meal. ABR has a large apparent distribution volume and it is highly bound to human plasma *Address correspondence to this author at the Department of Analytical Chemistry and Food Technology, University of Castilla La Mancha. Ciudad Real, Spain; Tel/Fax: (34)- 677364129; Fax: (34) 926-295318; E-mail:
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proteins (99.8%) [8]. After an oral administration of a dose, the recovery of the drug in feces and urine was 88% and 5% respectively. Major compounds in fecal samples are unchanged AA (55%) and ABR (22%). Time to reach maximum plasma ABR concentration is approximately 2h, the mean half-life of ABR in plasma is approximately 12h [5]. Although ABR has shown a good efficacy, responses are inconsistent over time. That could be due to the large interindividual variability in pharmacokinetics of ABR. Furthermore, ABR show clinical implications of drug–drug interactions through CYP3A4 metabolism pathway. As consequence, plasma and urine drug monitoring could be helpful in daily clinical practice for the management of outpatients treated with AA, because ABR therapy has been associated with increased levels of ALT, AST and bilirubin, prompting drug discontinuation or dosage modification [8].
A high performance liquid chromatography method with UV detection (HPLC-UV) has been described to assess abiraterone concentrations in rat plasma [9]. However, HPLCUV is not sensitive enough for drug monitoring in plasma of mCRPC patients. Recently, Bellevillle et al. [10] have reported an HPLC fluorescence method for abiraterone quantification in plasma. However, it requires a very timeconsuming stage for sample preparation. A liquid chromatographic-tandem mass spectroscopy (LC-MS/MS) method has also been reported for the analysis of ABR exposure in spike human plasma [11] and rat plasma [12]. The proposed method allows the rapid and simultaneous quantification of
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234 Current Pharmaceutical Analysis, 2018, Vol. 14, No. 3
Rodriguez et al.
AA and ABR in urine and serum of patients with mCRPC, using only one dilution step. Given the complex metabolism of AA, individual differences in bioavailability and the several adverse side-effects affecting most of the patients, it is necessary to develop new, faster and more sensitive methods for the evaluation of these drugs in biological fluids. This relevant information is the base for more effective and personalized therapies. Our previous investigation for breast cancer drugs aimed the same target [13]. N N
2.4. Sample Treatment The validation study was done on a mixture of three urines from different healthy men. Standard solution and internal standard were added to 1 mL of the urine mixture. Then mixture was vortexed for 1 min, diluted 1:10 (v/v) with the mobile phase corresponding to a mixture of acetonitrile: water 80:20 (v/v); then, vortexed again and centrifuged at 5000 x g for 3 min; a volume of 20 L of the supernatant was finally injected into the HPLC system. The serum samples were diluted 1:2 (v/v) with acetonitrile and centrifuged at 5000 x g for 3 min, a volume of 20 L of the supernatant was injected into the HPLC system.
CH3
3. RESULTS AND DISCUSSION
CH3
CH3
CH3
3.1. Optimization of Chromatographic Conditions
O
HO
O
(a)
(b)
Fig. (1). Chemical structures of (a) abiraterona acetate and (b) abiraterona.
2. MATERIALS AND METHODS 2.1. Reagents
AA was supplied by Santa Cruz Biotechnology (California), ABR by LGC Standards (Germany) and Progesterone (PGR) used as internal standard by Sigma. All solvents and reagents were of analytical grade unless otherwise indicated. Solutions were prepared in deionized water (Milli-Q quality).
Standard stock solution of ABR was prepared in ethanol at 520 mg/L, AA was prepared in DMSO at 1000mg/L and the internal standard PGR was prepared in methanol at 1000 mg/mL. The resulting solutions were stored at 4ºC. Ammonium acetate and HPLC-grade acetonitrile (SigmaAldrich) were used for the preparation of the mobile phase. The mobile phase was filtered through 0.45 m filters (HNWP membrane filters, Millipore). 2.2. Apparatus
A sample prepared as explained in the sample treatment section was used throughout the optimization process at drugs concentration of 10 ng/mL. For the HPLC fluorescence detection method, optimal excitation and emission wavelengths for the compounds were 255 and 370 nm respectively, for they present maximum intensity peaks at these wavelengths [6].
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CH3
A Shimadzu model LC-10AD HPLC coupled to a RF10AXL fluorescence and diode-array detector (Shimazdu, Kyoto, Japan) were used throughout the work. This equipment was fitted with a Rheodyne injection valve with a 20 L sample loop. The separation was carried out in a reverse phase in a Kromasil C8 column (150x4.6 mm). The system was controlled by Class-LC 10 software (Shimadzu, Kyoto, Japan), which was used for all measurements and data treatment. 2.3. Clinical Samples Clinical urine and serum samples from different men patients under Zytiga treatment were provided by the Oncology Department of the Hospital General de Ciudad Real (Spain). Urine and serum samples were collected in plastic containers and stored in the freezer at -18ºC until analysis.
The chromatographic parameters optimized for the separation of the analytes were pH, ionic strength and percentage of organic solvent in mobile phase; flow rate and temperature. Optimization was carried out by modifying the value of each parameter whilst keeping the rest constant, regarding sensitivity, peak resolution, and run time. As mobile phase, acetonitrile or methanol were tested as organic modifiers at different percentages, combined with acetic acetate and ammonium acetate buffers in the range of pH 4.2 to 7.7 and concentration between 20 and 60 mM. The optimal separation of the analytes was achieved with a mobile phase of acetonitrile with 20 mM ammonium acetate buffer (pH 7) in proportion 80:20 (v/v). Under these conditions, the effect of the chromatographic column temperature on the separation was studied between 18 and 35 ºC. A temperature of 30ºC provided the best resolution of all peaks in a short run time. For best separation the flow rate was studied from 0.8 to 1.4 mL/min. The best compromise among run time, separation, efficiency, peak width and column backpressure was obtained at 1.0 mL/min. As an example, the chromatograms in (Fig. 2) show a blank urine and urine spiked with 10 ng/mL of the two analytes. As can be seen, an excellent separation of the analytes was achieved in a run time of 11 min with no matrix interference, ABR and AA resulted in retention times of 5.1 min and 9.8 min respectively. The internal standard was recorded with diode array detector at 240 nm with a retention time of 3.5 min. 3.2. Validation of the Method 3.2.1. Linearity The analytical curves were constructed using a blank mixture of three urines spiked with standard solutions of ABR and AA in the range of 5.0 to 1100 ng/mL and PGR as internal standard at concentration of 3 g/mL which was determined with a diode array detector at 240 nm. The analytical curves were expressed in terms of relative area, the
A Rapid, Direct and Validated HPLC- Fluorescence Method A
Current Pharmaceutical Analysis, 2018, Vol. 14, No. 3 ABR
790
Fluorescence Intensity (mV)
235
690 590 490
AA
390 290 190 90 -10 0
2
4
6
8
10
12
Retention Time (min)
B
790 690 590 PGR
390
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muA
490
290 190 90
-10
0
2
4
6 Retention Time (min)
8
10
12
Fig. (2). A) Fluorescence chromatogram of a blank urine and chromatogram of a urine sample spiked at 10 ng/mL of the analytes and 3 g/mL of progesterone, (B) chromatogram at 240 nm. Table 1.
Precision.
Concentration ng/mL 12
500
1100
Compounds
Intra-Day Precision RSD (%), n=7
tR
Peak Area
Height
Relative
Relative
Inter-Day Precision RSD (%), n=2
tR
Peak area
Height
Relative
Relative
ABR
0.45
6.13
3.13
0.76
4.67
2.79
AA
0.49
5.85
3.21
0.81
4.13
3.56
ABR
0.18
3.31
2.22
0.19
2.59
1.89
AA
0.10
2.39
1.82
2.05
1.83
1.57
ABR
1.20
2.53
0.76
0.82
3.16
1.45
AA
0.88
2.59
1.02
0.73
3.02
1.42
regression equations of ABR and AA respectively were Y= (0.0017± 1.8910-5) X - (0.0035±0.0077) and Y= (0.0019±1.9810-5) X – (0.0003±0.0082). The correlation coefficients were 0.999 in both cases. 3.2.2. Limit of Detection and Quantitation The limits of detection (LOD), defined as the lowest concentration that the analytical process can reliably differentiate from background levels, were estimated for those concentrations that provide a signal-to-noise ratio of 3:1 which were found to be 6.9 ng/mL for ABR and 11.2 ng/mL for AA. The limits of quantification (LOQ) were estimated at a signal-to-noise ratio of 10:1 found to be 21 ng/mL for ABR and 34 ng/mL for AA.
3.2.3. Precision The precision of the chromatographic separation was evaluated by injecting a standard solution of all the analytes at 12, 500 and 1100 ng/mL under the optimized conditions seven times a day during two consecutive days. The results for intra-day and inter-day precision, in terms of relative standard deviation (RSD) of retention times and peak areas are shown in (Table 1). 3.2.4. Accuracy The accuracy of the method was expressed as relative error (%RE) (Table 2), which compares the mean observed quality control samples concentration with the theoretical
236 Current Pharmaceutical Analysis, 2018, Vol. 14, No. 3
Table 2.
Accuracy of chromatographic method. Concentration Obtained (ng/mL)
Concentration
Table 3.
Rodriguez et al.
RE (%)
Added (ng/mL)
ABR
AA
ABR
AA
12
11.20
11.51
-6.66
-4.08
500
511.52
501.55
2.30
0.31
1100
1082.29
1059.34
-1.61
-3.70
Recovery. Added (ng/mL)
%R
Added (ng/mL)
%R
Added (ng/mL)
%R
ABR
10
74.4
500
108.3
1000
104.8
AA
10
94.9
500
108.7
1000
107.8
Table 4.
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Sample Compounds
Analysis of human urine samples.
ABR (ng/mL) AA (ng/mL)
Man A
Man B
Man C
Man D
Man E
66.84
45.82
15.98
-
100.38
-
-
28.23
27.10
24.89
concentration (n=14), and is calculated with the following equation:
3.2.5. Stability
The stability of urine and serum samples in terms of retention time, peak area and height was investigated. The stability of urine and serum samples was studied injecting the sample every hour for 7 hours and the results revealed that it was stable during seven hours (RSD
