484 Original Article
Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies
Authors
M. Sasaki1, H. Ochiai1, K. Takahashi2, R. Suzuki1, K. Minato1, A. Fujikata1
Affiliations
1
Key words ▶ LC-MS/MS ● ▶ intramuscular administration ● ▶ vaginal administration ● ▶ ovariectomized rat ● ▶ pharmacokinetics ● ▶ steroids ●
Abstract
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389967 Published online: September 29, 2014 Drug Res 2015; 65: 484–489 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence M. Sasaki, PhD Pharmacokinetics Research Department Developmental Research Center ASKA Pharmaceutical Co., Ltd 5-36-1 Shimosakunobe Takatsu-ku Kawasaki 213-8522 Japan Tel.: + 81/44/812 8656 Fax: + 81/44/822 1265 [email protected]
Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan ASKA Pharma Medical Co., Ltd, Kawasaki, Japan
m/z 324.26 to m/z 113.07 for the IS, was used for quantification. Good linearity was observed over the concentration range of 0.05–20.00 ng/mL A sensitive liquid chromatography-tandem mass with a weighted (1/x2) linear regression. The spectrometry (LC-MS/MS) method was develintra- and inter-day precision ( % relative standoped and validated for the determination of proard deviation [RSD]) across 3 validation days over gesterone levels in rat plasma. Progesterone-d9 the entire concentration range was lower than was used as an internal standard (IS). Samples 6.7 %. Accuracy ( % nominal) determined at 5 were prepared using salting-out assisted liquid/ quality control concentrations was between 94.0 liquid extraction (SALLE), and the extracts were and 103.7 %. The validation method was applied injected directly onto the LC-MS/MS system. The in a pharmacokinetic study evaluating progesterchromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (100 mm × one levels after intramuscular or vaginal administration to ovariectomized (OVX) rats. The area 2.0 mm, i.d. 5 µm) using methanol and aqueous under the plasma concentration-time curve 0.1 % formic acid solution gradient as the mobile (AUC) calculated after intramuscular administraphase with a constant flow rate of 0.45 mL/min. tion was more than 4 times higher than the AUC Electrospray ionization in the positive-ion mode measured following vaginal administration of a was employed. Multiple reaction monitoring of comparable dose. the precursor to product ion pairs, from m/z 315.20 to m/z 109.10 for progesterone and from
▼
Introduction
▼
Progesterone is a natural steroid hormone secreted mainly by the mammalian ovary and placenta. It is administered intramuscularly or vaginally for luteal support in women undergoing in vitro fertilization-embryo transfer (IVF-ET) procedures [1, 2]. The pharmacokinetics of progesterone in humans have been well described in a number of studies. However, very few studies have evaluated the pharmacokinetics in experimental animals, despite the common use of rats in preclinical studies assessing the pharmacological and safety profiles of progesterone. We believe that it is important to clarify the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. Moreover, to avoid the confounding effect of menstrual cycle fluctuations in endogenous plasma progesterone levels, assessments need to be performed in ovariectomized (OVX) rats. To achieve this goal, it was necessary to develop a
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
novel analytical method capable of measuring progesterone concentrations below normal endogenous levels. Immunoassays, which have typically been used to determine progesterone levels, have poor selectivity due to their cross-reactivity. Although gas chromatography-mass spectrometry (GCMS) methods offer greater sensitivity and selectivity than immunoassays, such techniques generally require time-consuming derivatization steps. Recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods coupled with electrospray or atmospheric pressure chemical ionization make possible the development of novel highly reproducible, selective, and sensitive methods [3]. LC-MS/MS methods offer a powerful tool for the quantification of concentrations of nonvolatile and thermally labile compounds, usually without the need for derivatization. We, therefore, developed a novel method of measuring progesterone levels in rat plasma using LC-MS/MS.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
received 10.07.2014 accepted 01.09.2014
2
Original Article 485
Materials and Methods
▼
Materials
Progesterone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Progesterone-2,2,4,6,6,17α,21,21,21-d9 used as an internal standard (IS) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). Water was prepared from distilled water by using a Millipore Milli-Q water ultrapurification system (St. Louis, MO, USA). Special-grade acetonitrile, ethanol, and LC/MSgrade formic acid were purchased from Wako Pure Chemical (Osaka, Japan). Special-grade ammonium acetate was purchased from Nacalai Tesque (Kyoto, Japan). LC/MS-grade methanol was purchased from Kanto Chemical (Tokyo, Japan). Female rat control plasma was purchased from Charles River Laboratories (Kanagawa, Japan). Luteum® injection 10 (ASKA Pharma, Tokyo, Japan) was used as an intramuscular formulation. Vaginal suppositories containing 0.3 mg of progesterone were prepared in our laboratory by using Suppocire® (Hard Fat, GATTEFOSSE, Lyon, France).
Animals and ethical statement
Sprague-Dawley female rats (Crl:CD [SD], 5 weeks old) were purchased from Charles River Laboratories (Kanagawa, Japan). The rats were housed under a 12-h light/12-h dark cycle with controlled temperature (23 ± 1 °C) and humidity (50 ± 10 %). Food (FR-2, Funabashi Farm, Chiba, Japan) and water were provided ad libitum. The animals were allowed an acclimation period of 1 week before starting the experiment and ovariectomized under isoflurane anesthesia. The rats were administered progesterone within 2 weeks following the surgery. All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the institutional Animal Research Committee based on Rules for the Care and Use of Laboratory Animals in ASKA Pharmaceutical Co., Ltd.
Preparation of stock and working standard solutions
Stock solutions of progesterone and IS were prepared in ethanol at a concentration of 100 μg/mL and stored in a refrigerator. Separate stock solutions of progesterone were prepared for use as calibration standards and quality control samples. Working solutions of progesterone and a working solution of IS were prepared from the stock solutions by dilution with acetonitrile on each analysis day.
Sample preparation
A 50-μL aliquot of plasma was added to a 0.5-mL microcentrifuge tube containing 50-μL of IS working solution at a concentration of 5 ng/mL and 50-μL of acetonitrile. The tube was mixed vigorously for 10 s using a vortex-mixer. Following the addition of 50-μL of 5 mol/L ammonium acetate, tubes were vortexmixed for 10 s and centrifuged at 2 000 × g for 5 min. The top (organic) layer was transferred to an auto sampler vial and a 10-μL volume was injected onto the LC-MS/MS instrument.
Chromatographic and mass spectrometric conditions
The HPLC system (Shimadzu, Kyoto, Japan) consisted of a SCL10Avp system controller, 2 LC-20AD pumps, a SIL-HTc autosampler, and a CTO-20A column oven. The chromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (2.0 mm × 100 mm, i.d., 5 μm) (SHISEIDO, Tokyo, Japan). The mobile phase consisted of methanol and aqueous 0.1 % formic acid solution. A linear gradient elution was applied, according to the following profile (time [min]/methanol [ %]/flow [mL/min]): 0.00/57/0.45 → 1.00/57/0.45 → 5.50/80/0.45 → 5.51/98/0.45 → 5.52/98/0.45 → 5.53/98/0.70 → 6.50/98/0.70 → 6.51/57/0.70 → 6.52/57/0.70 → 6.53/57/0.60 → 8.00/57/0.60 → 8.01/57/0.45. An API5000 triple-quadrupole mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with TurboIonSpray source was operated in the positive ion multiple reaction monitoring mode for the analysis. The declustering potential was set to 136 V for progesterone and 110 V for IS. The collision energy and the collision cell exit potential were set at 39 eV and 18 eV, respectively. The mass transitions monitored for the determination of concentration of target compounds were as follows: for progesterone, from m/z 315.20 to m/z 109.10 and to m/z 97.00; and for IS, from m/z 324.26 to m/z 113.07. The quantitative product ion for progesterone was observed at m/z 109.10, while the m/z 97.00 corresponded to the qualitative product ion. Analyst 1.4.2 software (AB SCIEX, Foster city, CA, USA) was used to control the LC-MS/ MS system and perform sample data analyses.
Calibration curve
A 50-μL aliquot of water as a surrogate matrix was added to a 0.5-mL microcentrifuge tube containing 50-μL of IS working solution and 50-μL of each working solution to prepare calibration standards at concentrations of 0.05, 0.10, 0.25 0.50, 1.00, 5.00, 10.00, and 20.00 ng/mL. The calibration standards underwent the same extraction process as the plasma samples, as described in the previous subsection. A blank sample (processed surrogate matrix without progesterone or IS) and a zero sample (processed surrogate matrix with IS) were also prepared. The calibration curve was obtained by least-squares linear regression with a weighting factor of 1/x2 on the ratio of the peak area of progesterone to that of IS against the spiked concentrations in the calibration standards.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Sample preparations for bioanalysis have been conventionally performed using protein precipitation (PPT), liquid/liquid extraction (LLE), or solid phase extraction (SPE). Salting-out assisted liquid/liquid extraction (SALLE) was introduced for LC-MS analysis of biological samples and has a number of advantages over commonly used conventional methods [4]. Wu et al. reported the effective utilization of SALLE using mass-friendly salts as a sample preparation step prior to LC-MS analysis [5]. We selected ammonium acetate as a salting-out reagent and injected the SALLE extracts directly onto the LC-MS/MS instrument for the determination of progesterone levels in rat plasma. Development and validation of a quantitative method for measuring concentrations of endogenous compounds such as progesterone in a biological tissue sample is generally complicated by the presence of endogenous analytes in the matrix. Since endogenous progesterone was detectable in OVX rat plasma and in the charcoal-stripped rat plasma prepared in-house, water was used as the surrogate matrix for the preparation of the calibration standards. Our approach for bioanalytical method validation was to use the analyte-free surrogate matrix in combination with matrix-based quality control samples to evaluate the assay performance [6]. Our developed method was used for the determination of plasma progesterone concentrations after intramuscular or vaginal administration of progesterone to OVX rats.
486 Original Article Bioanalytical method validation
For the assessment of accuracy and precision, method validation runs that included a calibration curve, blank sample, zero sam-
ple, and quality control samples at 5 different concentrations were performed on 3 different days. Before the assessment of accuracy and precision, measurements were performed on
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Fig. 1 Multiple-reaction monitoring (MRM) chromatograms showing progesterone (left panel) and internal standard (IS; right panel) peaks detected using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) separation and analysis of a a blank sample, b 0.05 ng/mL calibration standard, c 5.00 ng/mL calibration standard, d pre-dose sample obtained from the OVX rat, and e postdose sample obtained 0.25 h after single vaginal administration of progesterone to the OVX rat.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Original Article 487 pooled control plasma (n = 10), and the mean concentration was used as the nominal baseline endogenous concentration. Quality control samples were prepared using pooled control plasma and surrogate matrix. An aliquot of pooled control matrix was used as an endogenous quality control sample (EQC). High quality control sample (HQC) was prepared by spiking the pooled control plasma with progesterone to the final concentration of 10 ng/mL, which is near the upper limit of quantification. Middle quality control sample (MQC) was prepared by diluting the pooled control plasma with water to the final concentration in the middle of the calibration range. Low quality control sample (LQC) was prepared by diluting the pooled control plasma with water to the final concentration within 3 times the lower limit of quantification (LLOQ). Surrogate matrix spiked with progesterone at the LLOQ was used as a quality control sample at the LLOQ
(LLQC). For the assessment of matrix effects, quality control samples at 4 different concentrations (LQC, MQC, EQC, and HQC) were prepared using 6 different lots of individual control plasma and surrogate matrix. All quality control samples were processed as described previously in this section.
Table 1 Calibration standards in 5 separate batches used for the validation of the LC-MS/MS quantification of progesterone levels in rat plasma.
The plasma concentrations of progesterone after intramuscular or vaginal administration to OVX rats were determined using the LC-MS/MS method in this study. Peak plasma concentration (Cmax) and the time to reach peak plasma concentrations (tmax) were determined from the plasma concentration-time curve. Pharmacokinetic parameters were estimated using the WinNonLin software package (Professional version 6.1; Pharsight, Mountain View, CA, USA). The area under the plasma concentration-time curve (AUC) was calculated by the linear/logarithmic trapezoidal rule and extrapolated to infinity by using the apparent terminal portion of the log plasma concentration-time curve. The apparent terminal half-life (t1/2) was estimated from the terminal rate constant as t1/2 = ln 2/λz, where λz is the terminal rate constant.
Deviation ( %) a
tration (ng/mL)
Batch 1
Batch 2
Batch 3
0.05 0.10 0.25 0.50 1.00 5.00 10.00 20.00 R R2
0.4 − 0.9 − 6.0 9.0 8.0 − 1.4 − 3.8 − 4.5 0.9980 0.9960
− 2.2 2.0 0.4 10.6 − 0.5 − 2.0 − 3.7 − 4.5 0.9985 0.9970
− 4.4 − 2.6 8.0 4.0 − 0.4 1.6 3.0 1.2 3.0 2.0 − 2.8 1.6 − 2.8 − 3.1 − 4.0 − 5.0 0.9988 0.9993 0.9976 0.9986
a
Batch 4
Batch 5 − 1.0 2.0 − 2.4 3.8 1.0 0.8 0.0 − 4.0 0.9996 0.9992
Pharmacokinetic experiments were performed in OVX rats. Rats were administered progesterone by intramuscular injection or as a vaginal suppository. Blood samples of approximately 100-μL in volume each were drawn from the tail vein at predefined time points up to 24 h using heparinized syringes. Plasma was obtained by centrifugation at 13 000 × g for 3 min. Samples were stored at − 20 °C until the day of the analysis.
Pharmacokinetics analysis
Deviation ( %) was calculated as (back-calculated concentration – spiked concentra-
tion/spiked concentration) × 100
Table 2 Intra-day and inter-day precision and accuracy of the measurements of progesterone in rat plasma. Batch
Day 1 (Batch 2)
Day 2 (Batch 3)
Day 3 (Batch 4)
Total
a
QC ID
LLOQ
LQC
MQC
EQC
HQC
Matrix
Surrogate matrix
Diluted matrix
Diluted matrix
Pooled control
Pooled control
(H2O)
(1:49)
(1:4)
matrix
matrix
Spiked (ng/mL) Nominal (ng/mL) a Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c
0.05 0.05 0.0511 0.0034 6.7 102.2 0.0487 0.0026 5.3 97.4 0.0501 0.0031 6.2 100.2 0.0500 0.0030 6.0 100.0
– 0.134 0.126 0.004 3.2 94.0 0.139 0.006 4.3 103.7 0.128 0.004 3.1 95.5 0.131 0.007 5.3 97.8
– 1.34 1.35 0.01 0.7 100.7 1.37 0.02 1.5 102.2 1.33 0.03 2.3 99.3 1.35 0.03 2.2 100.7
– 6.69 6.72 0.06 0.9 100.4 6.76 0.05 0.7 101.0 6.39 0.16 2.5 95.5 6.62 0.20 3.0 99.0
10.0 16.7 16.8 0.2 1.2 100.6 17.0 0.1 0.6 101.8 15.0 0.2 1.3 89.8 16.3 1.0 6.1 97.6
Nominal concentrations (ng/mL) were as follows:
Endogenous quality control sample (EQC): mean measured concentration of pooled control matrix (n = 10, Batch 1) High quality control sample (HQC): nominal concentration of EQC + spiked concentration Middle quality control sample (MQC) or low quality control sample (LQC): nominal concentration of EQC/dilution factor Quality control sample at the lower limit of quantification (LLQC): spiked concentration b c
Precision ( %) was calculated as (standard deviation [SD]/mean concentration) × 100
Accuracy ( %) was calculated as (mean concentration/nominal concentration) × 100
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Spiked concen-
Pharmacokinetic experiments in rats
488 Original Article
QC ID
LQC
MQC
EQC
HQC
plasma lot
Matrix
Diluted
Diluted
Matrix
Spiked matrix (10 ng/mL
matrix (1:49)
matrix (1:4)
1
Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b
0.114 0.107 93.9 0.188 0.167 88.8 0.182 0.171 94.0 0.122 0.112 91.8 0.0806 0.0756 93.8 0.128 0.119 93.0
1.14 1.15 100.9 1.88 1.91 101.6 1.82 1.85 101.6 1.22 1.24 101.6 0.806 0.815 101.1 1.28 1.30 101.6
2
3
4
5
6
a
spiked) – 5.72 – – 9.42 – – 9.11 – – 6.12 – – 4.03 – – 6.38 –
Table 3 Evaluation of the matrix effect in the measurement of progesterone in rat plasma.
15.7 15.1 96.2 19.4 19.4 100.0 19.1 19.0 99.5 16.1 15.7 97.5 14.0 13.8 98.6 16.4 15.8 96.3
Nominal concentrations (ng/mL) were as follows:
High quality control sample (HQC): found concentration of endogenous quality control sample (EQC) + spiked concentration Middle quality control sample (MQC) or low quality control sample (LQC): found concentration of EQC/dilution factor b
Analytical recovery ( %) was calculated as (found concentration/nominal concentration) × 100
Results
▼
Validation of the LC-MS/MS assay
Since progesterone was detectable even in the charcoal-stripped rat plasma prepared in-house, we chose water as the surrogate matrix for the preparation of calibration standards. Representative chromatograms obtained using the LC-MS/MS method are shown in ● ▶ Fig. 1. The linearity of the assay was assessed using the surrogate matrix calibration lines from each validation run. Good linearity was observed over the concentration range of 0.05–20.00 ng/mL with weighted (1/x2) linear regression. Relative errors were estimated between − 4.4 and 0.4 % at the LLOQ, and − 6.0 and 10.6 % for other concentrations, with correlation ▶ Table 1). coefficients greater than or equal to 0.9980 ( ● The approach for bioanalytical method validation was to use analyte-free surrogate matrix in combination with matrix-based QC samples to evaluate the assay performance. The results of intra-day and inter-day evaluation of accuracy and precision are summarized in ● ▶ Table 2. Precision ( % relative standard deviation [RSD]) values were within 6.7 % and accuracy ( % nominal) values were between 94.0 % and 103.7 %. These results met the acceptance criteria, indicating good reproducibility for the determination of progesterone levels in rat plasma and demonstrating water to be an adequate surrogate matrix for calibration standards. The results of matrix effect evaluation by using analyte-free surrogate matrix in combination with matrix-based QC samples are summarized in ● ▶ Table 3. The matrix effect was evaluated using analytical recovery, since the analyte is an endogenous compound detectable in blank plasma. Analytical recovery of measured analyte concentration in spiked and diluted matrix was between 88.8 % and 101.6 %, indicating that matrix effects were adequately controlled and the use of a surrogate matrix calibration was justified.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Table 4 The pharmacokinetic parameters calculated following intramuscular and vaginal administration of progesterone to OVX rats. administration Dose (mg/kg) Tmax (h) Cmax (ng/mL) AUC0–24 (ng · h/mL) AUC0–∞ (ng · h/mL) t1/2 (h)
Intramuscular 1.00 ± 0.00 2.00 ± 1.22 10.9 ± 1.6 91.2 ± 21.5 92.0 ± 22.0 3.03 ± 0.51
Vaginal 1.21 ± 0.13 0.63 ± 0.25 3.12 ± 0.53 22.1 ± 3.8 24.0 ± 5.4 5.55 ± 2.53
Data represent the means ± standard deviation (SD) of 4 (vaginal administration) or 5 (intramuscular administration) animals The dose used for vaginal administration is expressed normalized to body weight
Fig. 2 Plasma concentrations of progesterone measured after a single intramuscular (●, 1 mg/kg, n = 5) or vaginal (○, 0.3 mg/head, n = 4) administration of progesterone to OVX rats. Each point represents the mean ± standard deviation (SD).
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Individual
Original Article 489
Plasma concentrations of progesterone after intramuscular or vaginal administration to OVX rats were determined using the presented method. After an intramuscular progesterone administration (1 mg/kg), plasma levels reached Cmax (10.9 ng/mL) at 2 h, declined with t1/2 of 3.03 h, and decreased to basal levels by 24 h. The AUC0–24 and AUC0–∞ were 91.2 ng · h/mL and 92.0 ng · h/ mL, respectively. Following a vaginal administration (0.3 mg/ head), the plasma levels reached Cmax (3.12 ng/mL) at 0.63 h, declined with t1/2 of 5.55 h, and decreased to basal levels by 24 h. The AUC0–24 and AUC0–∞ were 22.1 ng · h/mL and 24.0 ng · h/mL, ▶ Table 4, ● ▶ Fig. 2). respectively ( ●
Discussion and Conclusion
▼
This work describes the development and validation of a highly sensitive LC-MS/MS method for the quantification of plasma progesterone concentrations in rats, using direct injection of SALLE extracts onto reversed-phase LC. Since the accuracy and precision of the presented analytical method satisfied the acceptance criteria and the matrix effect was adequately addressed, our findings show that the concentration of progesterone in rat plasma can be determined using water-based calibration standards at a concentration range between 0.05 and 20.00 ng/mL. We have applied the described method to evaluate the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. OVX rats were used to avoid the menstrual cycle fluctuations of endogenous plasma progesterone [7]. The metabolic clearance rate of progesterone was previously reported to be similar between OVX and normal rats [8]. Plasma concentrations of progesterone in OVX rats, measured before exogenous administration, were found to range between 0.074 and 0.406 ng/mL (data not shown), which is lower than the baseline progesterone levels observed in normal female rats (9–25 ng/ mL) [7]. Therefore, our study confirms that the concentrations of progesterone can be quantified at levels below the normal baseline. The plasma progesterone concentration in rats rapidly decreased after intravenous administration, with the disposition kinetics consistent with a 2-compartment model [9]. Considering these results, the elevated plasma levels of progesterone after intramuscular and vaginal administration may be maintained by the slow absorption from the site of administration (muscle or vaginal tissue). The AUC calculated after intramuscular administration was more than 4 times higher than the AUC obtained following vaginal administration at a comparable dose. Plasma concentration after oral administration of progesterone to rats was reported to decrease to one-third of Cmax within 10 min of dosing, with the bioavailability of the oral route calculated as 1.2 % [10]. In this study, intramuscular and vaginal administrations were confirmed to be useful routes that can sustain elevated plasma levels of progesterone in experimental animals.
We developed and validated a highly sensitive LC-MS/MS method for the quantification of plasma concentration of progesterone in rats, using direct injection of SALLE extracts onto the reversed-phase LC. The method has been successfully applied in a study assessing progesterone kinetics following intramuscular or vaginal administration in OVX rats. To our knowledge, this is the first report describing the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. Our results are likely to be useful to researchers developing progesterone applications using preclinical research involving animal models.
Acknowledgments
▼
We are grateful to K Shirota, H Aoki, H Nagao, M Kaneko, M Yoshizawa, Dr. K Bando, and S Fuse for their assistance with the animal experiments. We thank I Saito for providing the vaginal suppositories. All authors are employees of ASKA pharmaceutical Co., Ltd.
Conflict of Interest
▼
We declare that we have no conflict of interest.
References
1 Fatemi HM, Popovic-Todorovic B, Papanikolaou E et al. An update of luteal phase support in stimulated IVF cycles. Hum Reprod Update 2007; 13: 581–590 2 Tavaniotou A, Smitz J, Bourgain C et al. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update 2000; 6: 139–148 3 Tai SS, Xu B, Welch MJ. Development and evaluation of a candidate reference measurement procedure for the determination of progesterone in human serum using isotope-dilution liquid chromatography/ tandem mass spectrometry. Anal Chem 2006; 78: 6628–6633 4 Tang YQ, Weng N. Salting-out assisted liquid-liquid extraction for bioanalysis. Bioanalysis 2013; 5: 1583–1598 5 Wu H, Zhang J, Norem K et al. Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt. J Pharm Biomed Anal 2008; 48: 1243–1248 6 Houghton R, Pita CH, Ward I et al. Generic approach to validation of small-molecule LC-MS/MS biomarker assays. Bioanalysis 2009; 1: 1365–1374 7 Bonnamy PJ, Benhaim A, Leymarie P. Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle. J Reprod Fertil 1992; 96: 233–239 8 Pepe GJ, Rothchild I. Metabolic clearance rate of progesterone: Comparison between ovariectomized, pregnant, pseudopregnant and deciduoma-bearing pseudopregnant rats. Endocrinology 1973; 93: 1200–1205 9 Gangrade NK, Boudinot FD, Price JC. Pharmacokinetics of progesterone in ovariectomized rats after single dose intravenous administration. Biopharm Drug Dispos 1992; 13: 703–709 10 Bawarshi-Nassar RN, Hussain AA, Crooks PA. Nasal absorption and metabolism of progesterone and 17β-estradiol in the rat. Drug Metab Dispos 1989; 17: 248–254
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
The pharmacokinetics of progesterone in OVX rats
Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies
Authors
M. Sasaki1, H. Ochiai1, K. Takahashi2, R. Suzuki1, K. Minato1, A. Fujikata1
Affiliations
1
Key words ▶ LC-MS/MS ● ▶ intramuscular administration ● ▶ vaginal administration ● ▶ ovariectomized rat ● ▶ pharmacokinetics ● ▶ steroids ●
Abstract
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389967 Published online: September 29, 2014 Drug Res 2015; 65: 484–489 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence M. Sasaki, PhD Pharmacokinetics Research Department Developmental Research Center ASKA Pharmaceutical Co., Ltd 5-36-1 Shimosakunobe Takatsu-ku Kawasaki 213-8522 Japan Tel.: + 81/44/812 8656 Fax: + 81/44/822 1265 [email protected]
Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan ASKA Pharma Medical Co., Ltd, Kawasaki, Japan
m/z 324.26 to m/z 113.07 for the IS, was used for quantification. Good linearity was observed over the concentration range of 0.05–20.00 ng/mL A sensitive liquid chromatography-tandem mass with a weighted (1/x2) linear regression. The spectrometry (LC-MS/MS) method was develintra- and inter-day precision ( % relative standoped and validated for the determination of proard deviation [RSD]) across 3 validation days over gesterone levels in rat plasma. Progesterone-d9 the entire concentration range was lower than was used as an internal standard (IS). Samples 6.7 %. Accuracy ( % nominal) determined at 5 were prepared using salting-out assisted liquid/ quality control concentrations was between 94.0 liquid extraction (SALLE), and the extracts were and 103.7 %. The validation method was applied injected directly onto the LC-MS/MS system. The in a pharmacokinetic study evaluating progesterchromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (100 mm × one levels after intramuscular or vaginal administration to ovariectomized (OVX) rats. The area 2.0 mm, i.d. 5 µm) using methanol and aqueous under the plasma concentration-time curve 0.1 % formic acid solution gradient as the mobile (AUC) calculated after intramuscular administraphase with a constant flow rate of 0.45 mL/min. tion was more than 4 times higher than the AUC Electrospray ionization in the positive-ion mode measured following vaginal administration of a was employed. Multiple reaction monitoring of comparable dose. the precursor to product ion pairs, from m/z 315.20 to m/z 109.10 for progesterone and from
▼
Introduction
▼
Progesterone is a natural steroid hormone secreted mainly by the mammalian ovary and placenta. It is administered intramuscularly or vaginally for luteal support in women undergoing in vitro fertilization-embryo transfer (IVF-ET) procedures [1, 2]. The pharmacokinetics of progesterone in humans have been well described in a number of studies. However, very few studies have evaluated the pharmacokinetics in experimental animals, despite the common use of rats in preclinical studies assessing the pharmacological and safety profiles of progesterone. We believe that it is important to clarify the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. Moreover, to avoid the confounding effect of menstrual cycle fluctuations in endogenous plasma progesterone levels, assessments need to be performed in ovariectomized (OVX) rats. To achieve this goal, it was necessary to develop a
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
novel analytical method capable of measuring progesterone concentrations below normal endogenous levels. Immunoassays, which have typically been used to determine progesterone levels, have poor selectivity due to their cross-reactivity. Although gas chromatography-mass spectrometry (GCMS) methods offer greater sensitivity and selectivity than immunoassays, such techniques generally require time-consuming derivatization steps. Recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods coupled with electrospray or atmospheric pressure chemical ionization make possible the development of novel highly reproducible, selective, and sensitive methods [3]. LC-MS/MS methods offer a powerful tool for the quantification of concentrations of nonvolatile and thermally labile compounds, usually without the need for derivatization. We, therefore, developed a novel method of measuring progesterone levels in rat plasma using LC-MS/MS.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
received 10.07.2014 accepted 01.09.2014
2
Original Article 485
Materials and Methods
▼
Materials
Progesterone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Progesterone-2,2,4,6,6,17α,21,21,21-d9 used as an internal standard (IS) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). Water was prepared from distilled water by using a Millipore Milli-Q water ultrapurification system (St. Louis, MO, USA). Special-grade acetonitrile, ethanol, and LC/MSgrade formic acid were purchased from Wako Pure Chemical (Osaka, Japan). Special-grade ammonium acetate was purchased from Nacalai Tesque (Kyoto, Japan). LC/MS-grade methanol was purchased from Kanto Chemical (Tokyo, Japan). Female rat control plasma was purchased from Charles River Laboratories (Kanagawa, Japan). Luteum® injection 10 (ASKA Pharma, Tokyo, Japan) was used as an intramuscular formulation. Vaginal suppositories containing 0.3 mg of progesterone were prepared in our laboratory by using Suppocire® (Hard Fat, GATTEFOSSE, Lyon, France).
Animals and ethical statement
Sprague-Dawley female rats (Crl:CD [SD], 5 weeks old) were purchased from Charles River Laboratories (Kanagawa, Japan). The rats were housed under a 12-h light/12-h dark cycle with controlled temperature (23 ± 1 °C) and humidity (50 ± 10 %). Food (FR-2, Funabashi Farm, Chiba, Japan) and water were provided ad libitum. The animals were allowed an acclimation period of 1 week before starting the experiment and ovariectomized under isoflurane anesthesia. The rats were administered progesterone within 2 weeks following the surgery. All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the institutional Animal Research Committee based on Rules for the Care and Use of Laboratory Animals in ASKA Pharmaceutical Co., Ltd.
Preparation of stock and working standard solutions
Stock solutions of progesterone and IS were prepared in ethanol at a concentration of 100 μg/mL and stored in a refrigerator. Separate stock solutions of progesterone were prepared for use as calibration standards and quality control samples. Working solutions of progesterone and a working solution of IS were prepared from the stock solutions by dilution with acetonitrile on each analysis day.
Sample preparation
A 50-μL aliquot of plasma was added to a 0.5-mL microcentrifuge tube containing 50-μL of IS working solution at a concentration of 5 ng/mL and 50-μL of acetonitrile. The tube was mixed vigorously for 10 s using a vortex-mixer. Following the addition of 50-μL of 5 mol/L ammonium acetate, tubes were vortexmixed for 10 s and centrifuged at 2 000 × g for 5 min. The top (organic) layer was transferred to an auto sampler vial and a 10-μL volume was injected onto the LC-MS/MS instrument.
Chromatographic and mass spectrometric conditions
The HPLC system (Shimadzu, Kyoto, Japan) consisted of a SCL10Avp system controller, 2 LC-20AD pumps, a SIL-HTc autosampler, and a CTO-20A column oven. The chromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (2.0 mm × 100 mm, i.d., 5 μm) (SHISEIDO, Tokyo, Japan). The mobile phase consisted of methanol and aqueous 0.1 % formic acid solution. A linear gradient elution was applied, according to the following profile (time [min]/methanol [ %]/flow [mL/min]): 0.00/57/0.45 → 1.00/57/0.45 → 5.50/80/0.45 → 5.51/98/0.45 → 5.52/98/0.45 → 5.53/98/0.70 → 6.50/98/0.70 → 6.51/57/0.70 → 6.52/57/0.70 → 6.53/57/0.60 → 8.00/57/0.60 → 8.01/57/0.45. An API5000 triple-quadrupole mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with TurboIonSpray source was operated in the positive ion multiple reaction monitoring mode for the analysis. The declustering potential was set to 136 V for progesterone and 110 V for IS. The collision energy and the collision cell exit potential were set at 39 eV and 18 eV, respectively. The mass transitions monitored for the determination of concentration of target compounds were as follows: for progesterone, from m/z 315.20 to m/z 109.10 and to m/z 97.00; and for IS, from m/z 324.26 to m/z 113.07. The quantitative product ion for progesterone was observed at m/z 109.10, while the m/z 97.00 corresponded to the qualitative product ion. Analyst 1.4.2 software (AB SCIEX, Foster city, CA, USA) was used to control the LC-MS/ MS system and perform sample data analyses.
Calibration curve
A 50-μL aliquot of water as a surrogate matrix was added to a 0.5-mL microcentrifuge tube containing 50-μL of IS working solution and 50-μL of each working solution to prepare calibration standards at concentrations of 0.05, 0.10, 0.25 0.50, 1.00, 5.00, 10.00, and 20.00 ng/mL. The calibration standards underwent the same extraction process as the plasma samples, as described in the previous subsection. A blank sample (processed surrogate matrix without progesterone or IS) and a zero sample (processed surrogate matrix with IS) were also prepared. The calibration curve was obtained by least-squares linear regression with a weighting factor of 1/x2 on the ratio of the peak area of progesterone to that of IS against the spiked concentrations in the calibration standards.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Sample preparations for bioanalysis have been conventionally performed using protein precipitation (PPT), liquid/liquid extraction (LLE), or solid phase extraction (SPE). Salting-out assisted liquid/liquid extraction (SALLE) was introduced for LC-MS analysis of biological samples and has a number of advantages over commonly used conventional methods [4]. Wu et al. reported the effective utilization of SALLE using mass-friendly salts as a sample preparation step prior to LC-MS analysis [5]. We selected ammonium acetate as a salting-out reagent and injected the SALLE extracts directly onto the LC-MS/MS instrument for the determination of progesterone levels in rat plasma. Development and validation of a quantitative method for measuring concentrations of endogenous compounds such as progesterone in a biological tissue sample is generally complicated by the presence of endogenous analytes in the matrix. Since endogenous progesterone was detectable in OVX rat plasma and in the charcoal-stripped rat plasma prepared in-house, water was used as the surrogate matrix for the preparation of the calibration standards. Our approach for bioanalytical method validation was to use the analyte-free surrogate matrix in combination with matrix-based quality control samples to evaluate the assay performance [6]. Our developed method was used for the determination of plasma progesterone concentrations after intramuscular or vaginal administration of progesterone to OVX rats.
486 Original Article Bioanalytical method validation
For the assessment of accuracy and precision, method validation runs that included a calibration curve, blank sample, zero sam-
ple, and quality control samples at 5 different concentrations were performed on 3 different days. Before the assessment of accuracy and precision, measurements were performed on
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Fig. 1 Multiple-reaction monitoring (MRM) chromatograms showing progesterone (left panel) and internal standard (IS; right panel) peaks detected using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) separation and analysis of a a blank sample, b 0.05 ng/mL calibration standard, c 5.00 ng/mL calibration standard, d pre-dose sample obtained from the OVX rat, and e postdose sample obtained 0.25 h after single vaginal administration of progesterone to the OVX rat.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Original Article 487 pooled control plasma (n = 10), and the mean concentration was used as the nominal baseline endogenous concentration. Quality control samples were prepared using pooled control plasma and surrogate matrix. An aliquot of pooled control matrix was used as an endogenous quality control sample (EQC). High quality control sample (HQC) was prepared by spiking the pooled control plasma with progesterone to the final concentration of 10 ng/mL, which is near the upper limit of quantification. Middle quality control sample (MQC) was prepared by diluting the pooled control plasma with water to the final concentration in the middle of the calibration range. Low quality control sample (LQC) was prepared by diluting the pooled control plasma with water to the final concentration within 3 times the lower limit of quantification (LLOQ). Surrogate matrix spiked with progesterone at the LLOQ was used as a quality control sample at the LLOQ
(LLQC). For the assessment of matrix effects, quality control samples at 4 different concentrations (LQC, MQC, EQC, and HQC) were prepared using 6 different lots of individual control plasma and surrogate matrix. All quality control samples were processed as described previously in this section.
Table 1 Calibration standards in 5 separate batches used for the validation of the LC-MS/MS quantification of progesterone levels in rat plasma.
The plasma concentrations of progesterone after intramuscular or vaginal administration to OVX rats were determined using the LC-MS/MS method in this study. Peak plasma concentration (Cmax) and the time to reach peak plasma concentrations (tmax) were determined from the plasma concentration-time curve. Pharmacokinetic parameters were estimated using the WinNonLin software package (Professional version 6.1; Pharsight, Mountain View, CA, USA). The area under the plasma concentration-time curve (AUC) was calculated by the linear/logarithmic trapezoidal rule and extrapolated to infinity by using the apparent terminal portion of the log plasma concentration-time curve. The apparent terminal half-life (t1/2) was estimated from the terminal rate constant as t1/2 = ln 2/λz, where λz is the terminal rate constant.
Deviation ( %) a
tration (ng/mL)
Batch 1
Batch 2
Batch 3
0.05 0.10 0.25 0.50 1.00 5.00 10.00 20.00 R R2
0.4 − 0.9 − 6.0 9.0 8.0 − 1.4 − 3.8 − 4.5 0.9980 0.9960
− 2.2 2.0 0.4 10.6 − 0.5 − 2.0 − 3.7 − 4.5 0.9985 0.9970
− 4.4 − 2.6 8.0 4.0 − 0.4 1.6 3.0 1.2 3.0 2.0 − 2.8 1.6 − 2.8 − 3.1 − 4.0 − 5.0 0.9988 0.9993 0.9976 0.9986
a
Batch 4
Batch 5 − 1.0 2.0 − 2.4 3.8 1.0 0.8 0.0 − 4.0 0.9996 0.9992
Pharmacokinetic experiments were performed in OVX rats. Rats were administered progesterone by intramuscular injection or as a vaginal suppository. Blood samples of approximately 100-μL in volume each were drawn from the tail vein at predefined time points up to 24 h using heparinized syringes. Plasma was obtained by centrifugation at 13 000 × g for 3 min. Samples were stored at − 20 °C until the day of the analysis.
Pharmacokinetics analysis
Deviation ( %) was calculated as (back-calculated concentration – spiked concentra-
tion/spiked concentration) × 100
Table 2 Intra-day and inter-day precision and accuracy of the measurements of progesterone in rat plasma. Batch
Day 1 (Batch 2)
Day 2 (Batch 3)
Day 3 (Batch 4)
Total
a
QC ID
LLOQ
LQC
MQC
EQC
HQC
Matrix
Surrogate matrix
Diluted matrix
Diluted matrix
Pooled control
Pooled control
(H2O)
(1:49)
(1:4)
matrix
matrix
Spiked (ng/mL) Nominal (ng/mL) a Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c Mean (ng/mL) SD (ng/mL) Precision ( %) b Accuracy ( %) c
0.05 0.05 0.0511 0.0034 6.7 102.2 0.0487 0.0026 5.3 97.4 0.0501 0.0031 6.2 100.2 0.0500 0.0030 6.0 100.0
– 0.134 0.126 0.004 3.2 94.0 0.139 0.006 4.3 103.7 0.128 0.004 3.1 95.5 0.131 0.007 5.3 97.8
– 1.34 1.35 0.01 0.7 100.7 1.37 0.02 1.5 102.2 1.33 0.03 2.3 99.3 1.35 0.03 2.2 100.7
– 6.69 6.72 0.06 0.9 100.4 6.76 0.05 0.7 101.0 6.39 0.16 2.5 95.5 6.62 0.20 3.0 99.0
10.0 16.7 16.8 0.2 1.2 100.6 17.0 0.1 0.6 101.8 15.0 0.2 1.3 89.8 16.3 1.0 6.1 97.6
Nominal concentrations (ng/mL) were as follows:
Endogenous quality control sample (EQC): mean measured concentration of pooled control matrix (n = 10, Batch 1) High quality control sample (HQC): nominal concentration of EQC + spiked concentration Middle quality control sample (MQC) or low quality control sample (LQC): nominal concentration of EQC/dilution factor Quality control sample at the lower limit of quantification (LLQC): spiked concentration b c
Precision ( %) was calculated as (standard deviation [SD]/mean concentration) × 100
Accuracy ( %) was calculated as (mean concentration/nominal concentration) × 100
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Spiked concen-
Pharmacokinetic experiments in rats
488 Original Article
QC ID
LQC
MQC
EQC
HQC
plasma lot
Matrix
Diluted
Diluted
Matrix
Spiked matrix (10 ng/mL
matrix (1:49)
matrix (1:4)
1
Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b Nominal (ng/mL) a Found (ng/mL) Analytical recovery ( %) b
0.114 0.107 93.9 0.188 0.167 88.8 0.182 0.171 94.0 0.122 0.112 91.8 0.0806 0.0756 93.8 0.128 0.119 93.0
1.14 1.15 100.9 1.88 1.91 101.6 1.82 1.85 101.6 1.22 1.24 101.6 0.806 0.815 101.1 1.28 1.30 101.6
2
3
4
5
6
a
spiked) – 5.72 – – 9.42 – – 9.11 – – 6.12 – – 4.03 – – 6.38 –
Table 3 Evaluation of the matrix effect in the measurement of progesterone in rat plasma.
15.7 15.1 96.2 19.4 19.4 100.0 19.1 19.0 99.5 16.1 15.7 97.5 14.0 13.8 98.6 16.4 15.8 96.3
Nominal concentrations (ng/mL) were as follows:
High quality control sample (HQC): found concentration of endogenous quality control sample (EQC) + spiked concentration Middle quality control sample (MQC) or low quality control sample (LQC): found concentration of EQC/dilution factor b
Analytical recovery ( %) was calculated as (found concentration/nominal concentration) × 100
Results
▼
Validation of the LC-MS/MS assay
Since progesterone was detectable even in the charcoal-stripped rat plasma prepared in-house, we chose water as the surrogate matrix for the preparation of calibration standards. Representative chromatograms obtained using the LC-MS/MS method are shown in ● ▶ Fig. 1. The linearity of the assay was assessed using the surrogate matrix calibration lines from each validation run. Good linearity was observed over the concentration range of 0.05–20.00 ng/mL with weighted (1/x2) linear regression. Relative errors were estimated between − 4.4 and 0.4 % at the LLOQ, and − 6.0 and 10.6 % for other concentrations, with correlation ▶ Table 1). coefficients greater than or equal to 0.9980 ( ● The approach for bioanalytical method validation was to use analyte-free surrogate matrix in combination with matrix-based QC samples to evaluate the assay performance. The results of intra-day and inter-day evaluation of accuracy and precision are summarized in ● ▶ Table 2. Precision ( % relative standard deviation [RSD]) values were within 6.7 % and accuracy ( % nominal) values were between 94.0 % and 103.7 %. These results met the acceptance criteria, indicating good reproducibility for the determination of progesterone levels in rat plasma and demonstrating water to be an adequate surrogate matrix for calibration standards. The results of matrix effect evaluation by using analyte-free surrogate matrix in combination with matrix-based QC samples are summarized in ● ▶ Table 3. The matrix effect was evaluated using analytical recovery, since the analyte is an endogenous compound detectable in blank plasma. Analytical recovery of measured analyte concentration in spiked and diluted matrix was between 88.8 % and 101.6 %, indicating that matrix effects were adequately controlled and the use of a surrogate matrix calibration was justified.
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Table 4 The pharmacokinetic parameters calculated following intramuscular and vaginal administration of progesterone to OVX rats. administration Dose (mg/kg) Tmax (h) Cmax (ng/mL) AUC0–24 (ng · h/mL) AUC0–∞ (ng · h/mL) t1/2 (h)
Intramuscular 1.00 ± 0.00 2.00 ± 1.22 10.9 ± 1.6 91.2 ± 21.5 92.0 ± 22.0 3.03 ± 0.51
Vaginal 1.21 ± 0.13 0.63 ± 0.25 3.12 ± 0.53 22.1 ± 3.8 24.0 ± 5.4 5.55 ± 2.53
Data represent the means ± standard deviation (SD) of 4 (vaginal administration) or 5 (intramuscular administration) animals The dose used for vaginal administration is expressed normalized to body weight
Fig. 2 Plasma concentrations of progesterone measured after a single intramuscular (●, 1 mg/kg, n = 5) or vaginal (○, 0.3 mg/head, n = 4) administration of progesterone to OVX rats. Each point represents the mean ± standard deviation (SD).
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Individual
Original Article 489
Plasma concentrations of progesterone after intramuscular or vaginal administration to OVX rats were determined using the presented method. After an intramuscular progesterone administration (1 mg/kg), plasma levels reached Cmax (10.9 ng/mL) at 2 h, declined with t1/2 of 3.03 h, and decreased to basal levels by 24 h. The AUC0–24 and AUC0–∞ were 91.2 ng · h/mL and 92.0 ng · h/ mL, respectively. Following a vaginal administration (0.3 mg/ head), the plasma levels reached Cmax (3.12 ng/mL) at 0.63 h, declined with t1/2 of 5.55 h, and decreased to basal levels by 24 h. The AUC0–24 and AUC0–∞ were 22.1 ng · h/mL and 24.0 ng · h/mL, ▶ Table 4, ● ▶ Fig. 2). respectively ( ●
Discussion and Conclusion
▼
This work describes the development and validation of a highly sensitive LC-MS/MS method for the quantification of plasma progesterone concentrations in rats, using direct injection of SALLE extracts onto reversed-phase LC. Since the accuracy and precision of the presented analytical method satisfied the acceptance criteria and the matrix effect was adequately addressed, our findings show that the concentration of progesterone in rat plasma can be determined using water-based calibration standards at a concentration range between 0.05 and 20.00 ng/mL. We have applied the described method to evaluate the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. OVX rats were used to avoid the menstrual cycle fluctuations of endogenous plasma progesterone [7]. The metabolic clearance rate of progesterone was previously reported to be similar between OVX and normal rats [8]. Plasma concentrations of progesterone in OVX rats, measured before exogenous administration, were found to range between 0.074 and 0.406 ng/mL (data not shown), which is lower than the baseline progesterone levels observed in normal female rats (9–25 ng/ mL) [7]. Therefore, our study confirms that the concentrations of progesterone can be quantified at levels below the normal baseline. The plasma progesterone concentration in rats rapidly decreased after intravenous administration, with the disposition kinetics consistent with a 2-compartment model [9]. Considering these results, the elevated plasma levels of progesterone after intramuscular and vaginal administration may be maintained by the slow absorption from the site of administration (muscle or vaginal tissue). The AUC calculated after intramuscular administration was more than 4 times higher than the AUC obtained following vaginal administration at a comparable dose. Plasma concentration after oral administration of progesterone to rats was reported to decrease to one-third of Cmax within 10 min of dosing, with the bioavailability of the oral route calculated as 1.2 % [10]. In this study, intramuscular and vaginal administrations were confirmed to be useful routes that can sustain elevated plasma levels of progesterone in experimental animals.
We developed and validated a highly sensitive LC-MS/MS method for the quantification of plasma concentration of progesterone in rats, using direct injection of SALLE extracts onto the reversed-phase LC. The method has been successfully applied in a study assessing progesterone kinetics following intramuscular or vaginal administration in OVX rats. To our knowledge, this is the first report describing the pharmacokinetics of progesterone in rats following administration by clinically relevant routes. Our results are likely to be useful to researchers developing progesterone applications using preclinical research involving animal models.
Acknowledgments
▼
We are grateful to K Shirota, H Aoki, H Nagao, M Kaneko, M Yoshizawa, Dr. K Bando, and S Fuse for their assistance with the animal experiments. We thank I Saito for providing the vaginal suppositories. All authors are employees of ASKA pharmaceutical Co., Ltd.
Conflict of Interest
▼
We declare that we have no conflict of interest.
References
1 Fatemi HM, Popovic-Todorovic B, Papanikolaou E et al. An update of luteal phase support in stimulated IVF cycles. Hum Reprod Update 2007; 13: 581–590 2 Tavaniotou A, Smitz J, Bourgain C et al. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update 2000; 6: 139–148 3 Tai SS, Xu B, Welch MJ. Development and evaluation of a candidate reference measurement procedure for the determination of progesterone in human serum using isotope-dilution liquid chromatography/ tandem mass spectrometry. Anal Chem 2006; 78: 6628–6633 4 Tang YQ, Weng N. Salting-out assisted liquid-liquid extraction for bioanalysis. Bioanalysis 2013; 5: 1583–1598 5 Wu H, Zhang J, Norem K et al. Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt. J Pharm Biomed Anal 2008; 48: 1243–1248 6 Houghton R, Pita CH, Ward I et al. Generic approach to validation of small-molecule LC-MS/MS biomarker assays. Bioanalysis 2009; 1: 1365–1374 7 Bonnamy PJ, Benhaim A, Leymarie P. Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle. J Reprod Fertil 1992; 96: 233–239 8 Pepe GJ, Rothchild I. Metabolic clearance rate of progesterone: Comparison between ovariectomized, pregnant, pseudopregnant and deciduoma-bearing pseudopregnant rats. Endocrinology 1973; 93: 1200–1205 9 Gangrade NK, Boudinot FD, Price JC. Pharmacokinetics of progesterone in ovariectomized rats after single dose intravenous administration. Biopharm Drug Dispos 1992; 13: 703–709 10 Bawarshi-Nassar RN, Hussain AA, Crooks PA. Nasal absorption and metabolism of progesterone and 17β-estradiol in the rat. Drug Metab Dispos 1989; 17: 248–254
Sasaki M et al. PK of Progesterone in OVX Rats … Drug Res 2015; 65: 484–489
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
The pharmacokinetics of progesterone in OVX rats
