MS: Application to a bioequivalence study in healthy volunteers

Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 213–217

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Analysis of 21-hydroxy deflazacort in human plasma by UPLC–MS/MS: Application to a bioequivalence study in healthy volunteers Daxesh P. Patel a , Primal Sharma a , Bhargav M. Patel a , Mallika Sanyal b , Puran Singhal c , Pranav S. Shrivastav a,∗ a

Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, India Department of Chemistry, St. Xavier’s College, Navrangpura, Ahmedabad 380009, India c Bioanalytical Department, Alkem Laboratories Ltd., Lower Parel, Mumbai 400013, India b

a r t i c l e

i n f o

Article history: Received 13 March 2013 Received in revised form 19 July 2013 Accepted 20 July 2013 Available online 6 August 2013 Keywords: 21-Hydroxy deflazacort UPLC–MS/MS Sensitive Bioequivalence study Incurred sample reanalysis

a b s t r a c t A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method has been developed for the determination of 21-hydroxy deflazacort in human plasma using betamethasone as the internal standard (IS). After solid-phase extraction from 100 ␮L human plasma, the analyte and IS were analyzed on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 ␮m) column using acetonitrile-4.0 mM ammonium formate, pH 3.5 (90:10, v/v) as the mobile phase. The protonated analyte was quantified by selected reaction monitoring in the positive ionization mode by triple quadrupole mass spectrometer. The calibration plots were linear over the concentration range 0.50–500 ng/mL. Intra-batch and inter-batch precision (% CV) and accuracy (%) for five quality control samples ranged within 1.40–4.82% and 98.0–102.0% respectively. The overall mean extraction recovery of 21-hydroxy deflazacort from plasma ranged from 95.3 to 97.3%. Matrix effect was assessed by postcolumn analyte infusion and the extraction recovery was >95.0% across four quality control levels for the analyte and IS. Stability was evaluated under different conditions like bench top, autosampler, processed sample (at room temperature and in cooling chamber), freeze-thaw and long term stability. The method was applied to support a bioequivalence study of 30 mg deflazacort tablet formulation in 28 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 115 incurred samples. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Deflazacort (DFZ) is a synthetic glucocorticoid with antiinflammatory and immunosuppressant activity. It is a methyloxazolone derivative of prednisolone with high affinity for type II glucocorticoid receptors. The drug is prescribed for the treatment of several disorders, including rheumatoid arthritis, asthama, Duchenne muscular dystrophy and ureterolithiasis [1,2]. DFZ is a prodrug and gets rapidly absorbed and metabolized by plasma esterases to its pharmacologically active metabolite 21-hydroxy deflazacort (21-OH DFZ). The plasma concentration of DFZ after oral administration are very low, while 21-OH DFZ can be readily quantified with peak plasma concentration between 1 and 2 h. The plasma elimination half life of the metabolite is about 1.5–2 h and is approximately 40% protein bound [3,4].

∗ Corresponding author. Tel.: +91 079 26300969; fax: +91 079 26308545; Mobile: +91 9925471963. E-mail address: pranav [email protected] (P.S. Shrivastav). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.035

Several methods are reported for the determination of 21-OH DFZ by HPLC [3–8] and LC–MS/MS [9] in biological fluids. Bulk of these methods have limitations of low sensitivity (>1 ng/mL) [3–7], high sample volume for processing (≥0.5 mL) [3–8] and long chromatographic analysis time (>5 min) [3–8]. Developed procedures for plasma extraction of 21-OH DFZ have used liquid–liquid extraction (LLE) [5,9] and solid phase extraction (SPE) [8]. Ifa and coworkers [9] have described a rapid LC–MS/MS method with a linear dynamic range from 1.0 to 400 ng/mL for 21-OH DFZ in human plasma. They used diethyl ether–dichloromethane as extraction solvent to obtain recovery in the range of 68–71%. Reynolds et al. [8] developed a gradient semi-microbore liquid chromatography method for sensitive determination (1.0 ng/mL) of 21-OH DFZ. They employed a solid-phase extraction (SPE) procedure using C18 cartridges for precise and quantitative recovery (78.1–85.8%) of 21-OH DFZ from human plasma. Nevertheless, due to sensitivity requirement large plasma volume (2.0 mL) was required for processing in their work. Moreover, sample cleanup during elution step was dependent on the pH of the solid-phase material used and washing with an acidic buffer was essential to prevent co-elution of interfering matrix components. Salient features of methods developed for

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Table 1 Salient features of methods developed for determination of 21-hydroxy deflazacort in biological samples. Sr. no.

Detection technique

Sample volume

Extraction procedure; internal standard

Recovery (%)

1a

HPLC-UV

500/1000 ␮L human plasma

91–103

2b

HPLC-UV

1000 ␮L human serum

3c

HPLC-UV

3000 ␮L human urine

4

HPLC-UV

2000 ␮L human plasma

LLE with methylene chloride; prednisolone SPE on Sep-Pak C18 Waters cartridges; betamethasone LLE with dichloromethane; methyl prednisolone SPE on Clean-Up C18 cartridges; fludrocortisone

5

LC–MS/MS

200 ␮L human plasma

6

UPLC–MS/MS

100 ␮L human plasma

a b c

LLE with diethyl ether–dichloromethane; 21-acetate dexamethazone SPE on Phenomenex Strata cartridges; betamethasone

Run time

Application

Ref.

10–600

10 min

[5]

91.3–93.8

10–300

40 min

91.0–93.4

130–660

18 min

78.1–85.8

1.0–500

14 min

68–71

1.0–400

5 min

Pharmacokinetic study with 60, 30, 18 and 6 mg DFZ in 12 healthy subjects Pharmacokinetic study with DFZ in healthy volunteers Pharmacokinetic study with 30 mg DFZ in healthy volunteers Pharmacokinetic study with 6.0 mg DFZ in healthy subjects Bioequivalence study with 30 mg DFZ in 24 healthy subjects Bioequivalence study with 30 mg DFZ in 28 healthy subjects

95.3–97.3

Linear range (ng/mL)

0.50–500

1.5 min

[6]

[7]

[8]

[9]

PM

Along with another metabolite. Along with another metabolite, cortisol, cortisone, prednisone and prednisolone. Along with DFZ; DFZ: deflazacort; LLE: liquid–liquid extraction; SPE: solid phase extraction; PM: present method.

21-OH DFZ are summarized in Table 1. To the best of our knowledge there are no reports on the use of UPLC–MS/MS for determination of 21-OH DFZ in human plasma. Additionally, pharmacokinetic data for Indian subjects is not available in the literature [10]. Ultra performance liquid chromatography (UPLC) is an ideal tool for rapid separation of complex mixtures in both isocratic and gradient modes. Improved separation efficiency and a decrease in the analysis time can be realized by reducing the particle size of the stationary phase. The advantage of UPLC over conventional HPLC is the ability to increase the speed without sacrificing efficiency [11]. In the present work a sensitive, selective and rapid UPLC–MS/MS method has been developed and fully validated for reliable measurement of 21-OH DFZ in human plasma. The method requires only 100 ␮L human plasma for sample processing and demonstrates superior chromatographic performance in a run time of 1.5 min. It was applied to support a bioequivalence study in 28 healthy subjects. 2. Experimental

Quattro Premier XETM mass spectrometer from Waters–Micro Mass Technologies (MA, USA), in the positive electro spray ionization mode. Mass parameters were suitably optimized to obtain a consistent and sufficient response for 21-OH DFZ and IS (Supplementary Table S1). A dwell time of 50 ms gave a minimum of 27 data points across the peaks for quantitative reproducibility and sensitivity. MassLynx software version 4.1 was used to control all parameters of UPLC and MS.

2.3. Calibrators and quality control samples Calibration standards (CSs) were made at 0.500, 1.00, 3.00, 10.0, 30.0, 50.0, 100, 200, 350, 500 ng/mL concentrations. Five quality control samples were prepared (a) 425 ng/mL (HQC, high quality control), (b) 250/20.0 ng/mL (MQC-1/2, medium quality control), (c) 1.50 ng/mL (LQC, low quality control) and (d) 0.500 ng/mL (LLOQ QC, lower limit of quantification quality control). The details of solution preparation are provided in Appendix A – Supplementary Data.

2.1. Chemicals and materials The details of reference standards, their purity and other materials used in the study are given in Appendix A – Supplementary Data. 2.2. Liquid chromatographic and mass spectrometric conditions The chromatographic analysis of 21-OH DFZ and IS was carried out on Waters Acquity UPLC system (MA, USA) with UPLC BEH C18 (50 mm × 2.1 mm, 1.7 ␮m) analytical column, maintained at 30 ◦ C. The mobile phase consisted of acetonitrile-4.0 mM ammonium formate, pH 3.5, adjusted with formic acid (90:10, v/v) and was delivered at a flow rate of 0.250 mL/min. The sample manager temperature was maintained at 5 ◦ C and the pressure of the system was 6600 psi. Quantitation was done using selected reaction monitoring (SRM) for protonated precursor → product ion transitions on

2.4. Protocol for sample preparation Prior to analysis, all frozen subject samples, calibration standards and quality control samples were thawed and allowed to equilibrate at room temperature for 30 min. To an aliquot of 100 ␮L of spiked plasma sample, 30 ␮L of internal standard was added and vortexed for 10 s. Thereafter, the samples were centrifuged at 13,148 × g for 5 min at 10 ◦ C. Plasma samples were then applied to Phenomenex Strata-X (30 mg, 1 cc) cartridges, which were preconditioned with 1 mL methanol followed by 1 mL of water. The samples were washed twice with 1 mL, 5% methanol in water. Drying of cartridges was done for 1 min by applying nitrogen (1.72 × 105 Pa) at 2.4 L/min flow rate. The analyte and IS were eluted with 300 ␮L mobile phase into pre-labeled vials, vortexed for 15 s and 10 ␮L was used for injection in the chromatographic system.

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Fig. 1. Representative SRM ion-chromatograms of (a) double blank plasma (without analyte and IS), (b) blank plasma with working solution of betamethasone (m/z 393.1 → 147.0), IS (c) 21-hydroxy deflazacort at LLOQ (m/z 400.2 → 124.1) and IS, (d) 21-hydroxy deflazacort in subject sample at Cmax and IS after oral administration of Deflanil, 30 mg deflazacort tablet.

2.5. Procedures for method validation The method validation was performed as per the USFDA guidelines [12]. Details of validation procedure and acceptance criteria are given in Appendix A – Supplementary data.

2.6. Bioequivalence study design and incurred sample reanalysis The bioequivalence study was conducted with a single dose of 30 mg Deflanil (Libbs Farmaceutica Ltd., Sao Paulo, Brazil) and 30 mg Calcort (Sanofi-Aventis Farmaceutica Ltd., Brazil) deflazacort tablet formulations in 28 healthy adult Indian subjects under fasting conditions. The study was conducted as per International Conference on Harmonization, E6 Good Clinical Practice guidelines [13]. Incurred sample reanalysis was performed as reported in a previous report [14]. The details for both the experiments are provided in Appendix A – Supplementary data.

3. Results and discussion 3.1. Method development Mass spectrometric conditions were suitably optimized to obtain maximum sensitivity for 21-OH DFZ and betamethasone (IS). The Q1 mass full scan spectra contained protonated precursor [M+H]+ ions at m/z 400.2 for 21-OH DFZ and m/z 393.1 for IS. The most stable and abundant product ions in Q3 mass spectra were observed at m/z 124.1 and 147.0 for 21-OH DFZ and IS respectively. The ion at m/z 124.1 for 21-OH DFZ can be ascribed to the fragment having the oxazole group and devoid of a water molecule (Supplementary Fig. S1a) as reported previously [9]. The fragmentation pathway for IS was largely dependent on collision energy as reported by Polettini et al. [15]. At low collision energy (10–15 eV) several product ions at m/z 373.2, 355.1 and 337.1 were observed, which corresponded to the elimination of HF, HF + H2 O and HF + 2H2 O respectively from the protonated precursor ions. However, a consistent and abundant product ion at m/z 147.0 was found by applying 40 eV collision energy (Supplementary Fig. S1b). In the present work, SPE was carried out on Phenomenex StrataTM -X (30 mg, 1 cc), which required minimal steps for sample cleanup and ensured quantitative and precise recovery at all QC levels for the analyte and IS (Table 2). Washing of cartridges with

5% methanol was adequate for complete removal of interfering compounds. Chromatographic conditions were suitably optimized under isocratic conditions to get adequate response, acceptable peak shape and a short analysis time on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 ␮m) column. Various combinations of organic diluents (methanol/acetonitrile) together with acidic buffers (ammonium formate/formic acid, ammonium acetate/acetic acid) with different ionic strengths (2–8 mM) in the pH range of 3.5–5.5 were tested. The best mobile phase system which afforded adequate retention and peak shape was acetonitrile-4.0 mM ammonium formate, pH 3.5, adjusted with formic acid (90:10, v/v). The analyte and IS were eluted within 1.5 min with retention times of 1.05 and 1.11 min respectively. Betamethasone, a synthetic glucocorticoid having very similar structure was used as an internal standard. It adequately compensated for any variability during sample extraction and MS ionization. Representative SRM ion chromatograms in Fig. 1a–d verify the selectivity of the method to differentiate and quantify the analyte from endogenous components in the plasma matrix. The post column infusion experiment showed no interfering signal at the retention time of the analyte and IS (Fig. 2). Further, none of the commonly used medications by human volunteers interfered at the retention of 21-OH DFZ. Absolute matrix effect values varied from 100.8 to 102.9% indicating a minor ion enhancement (Table 2). The % CV value calculated for relative matrix effect in different plasma sources was 2.81, which is within the acceptance criteria of ≤3.0% (Supplementary Table S2).

Fig. 2. Injection of extracted blank human plasma during post column infusion of (a) 21-hydroxy deflazacort at HQC level and (b) betamethasone (IS).

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Table 2 Recovery and matrix effect data for 21-hydroxy deflazacort (n = 6). Mean area response

A: Neat standards (% CV)

B: Post-extraction fortified samples (% CV)

C: Pre-extraction fortified samples (% CV)

Low quality control 1155 (3.17)

Absolute matrix effect, B/A × 100 21-OH DFZ (IS)

Relative recovery, C/B × 100 21-OH DFZ (IS)

Process efficiency, C/A × 100 21-OH DFZ (IS)

1170 (2.81)

1116 (2.88)

101.3 (97.0)

95.4 (95.2)

96.6 (92.3)

Medium quality control-2 15,511 (1.64)

15,650 (1.70)

15,178 (2.49)

100.9 (96.2)

96.9 (96.9)

97.8 (93.2)

Medium quality control-1 194435 (2.89)

195952 (2.88)

190661 (1.56)

100.8 (95.2)

97.3 (96.7)

98.0 (92.1)

High quality control 328,458 (2.57)

337,983 (2.58)

322,098 (2.97)

102.9 (96.0)

95.3 (96.8)

98.1 (92.9)

3.2. Assay results The precision (% CV) values for system suitability test varied from 0.08 to 0.21% for the retention time and 0.54–0.83% for the area response of 21-OH DFZ and IS. The signal to noise ratio for system performance was ≥20. The auto-sample carryover experiment showed minimal carryover of analyte, ≤0.07% of LLOQ area in the extracted blank sample after injection of ULOQ sample. The calibration curves showed good linearity (r2 ≥ 0.9996) in the studied concentration range of 0.50–500 ng/mL for 21-OH DFZ. The mean linear equation for calibration curve concentrations was y = (0.00401 ± 0.00010) x + (0.00050 ± 0.00003). The accuracy (%) and precision (% CV) values for CSs ranged from 98.9 to 101.6% and 0.52 to 2.06% respectively. The lower limit of quantitation (0.50 ng/mL) was measured at a signal-to-noise ratio (S/N) ≥ 20. The intra-batch precision (% CV) varied from 1.40 to 3.71 while the accuracy was within 98.8–102.0%. For the inter-batch experiments, the precision (% CV) ranged from 2.04 to 4.82 and the accuracy was between 98.0 and 101.9% (Supplementary Table S3). The short-term and long term stock solution stability of analyte and IS remained unchanged up to 28 h and for 29 days respectively with a % change ≤1.5. The detailed results of analyte stability in plasma are shown in Table 3. The precision (% CV) and accuracy values on different columns and analysts for method ruggedness ranged from 1.2 to 3.1% and 97.8 to 102.4% respectively across five QC levels. The precision values for dilution reliability with 1/5th and 1/10th dilution were 1.2 and 1.7%, while the corresponding accuracy results were 97.5 and 102.5% respectively.

Fig. 3. Mean plasma concentration-time profile of Deflanil (30 mg deflazacort tablet, Libbs Farmaceutica Ltd., Sao Paulo, Brazil) and Calcort (30 mg deflazacort tablet, Sanofi-Aventis Farmaceutica Ltd., Brazil) after oral administration to 28 healthy subjects.

3.3. Application of the method in healthy subjects and incurred sample results So far there are no reports on the pharmacokinetics of 21-OH DFZ in Indian subjects. Thus, a preliminary study was done with 28 healthy Indian males using 30 mg deflazacort tablets. Fig. 3 shows the plasma concentration vs. time profile of 21-OH DFZ in healthy volunteers under fasting condition. The Cmax , AUC0−t and AUC0−inf values obtained were higher compared to a previous study with similar formulations and identical dose strength [9]. This

Table 3 Stability of 21-hydroxy deflazacort in plasma under various conditions (n = 6). Storage conditions Bench top stability at room temperature for 20 h ◦

Freeze and thaw stability, 5th cycle at −20 C Freeze and thaw stability, 5th cycle at −70 ◦ C Autosampler stability at 4 ◦ C, 98 h Processed sample stability at room temperature (16 h) Processed sample stability in cooling chamber at 5 ◦ C, 30 h Long term stability at −20 ◦ C, 176 days Long term stability at −70 ◦ C, 176 days

Nominal concentration (ng/mL)

Mean stability sample (ng/mL) ± SD

HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC LQC

431 1.52 429 1.48 420 1.53 430 1.51 423 1.47 427 1.49 428 1.48 422 1.53

425 1.50 425 1.50 425 1.50 425 1.50 425 1.50 425 1.50 425 1.50 425 1.50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.97 0.04 10.7 0.05 9.89 0.04 6.89 0.04 8.75 0.04 8.46 0.04 7.21 0.05 10.3 0.04

% Change 1.40 1.32 0.94 −1.34 −1.18 2.00 1.16 0.67 −0.47 −2.00 0.47 −0.67 0.71 −1.33 −0.70 2.00

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difference could be ascribed to ethnicity, gender, food and other factors. However, Tmax and t1/2 values for 21-OH DFZ were nearly identical with their study. Supplementary Table S4 summarizes the mean pharmacokinetic parameters after oral administration of 30 mg deflazacort (Deflanil and Calcort) tablet formulations. No significant difference was found between the two formulations in any parameter. The ratios of mean log-transformed parameters (Cmax , AUC0−t , and AUC0−inf ) and their 90% CIs were all within the defined bioequivalence range of 80–125%. Both formulations were well tolerated in healthy volunteers and there was no adverse event during the course of the study. This confirms the bioequivalence of the two formulations in terms of rate and extent of absorption. About 2100 samples were analyzed with the proposed method during a period of 7 days with acceptable precision and accuracy. Further, the reproducibility of the method was confirmed by reanalysis of 115 incurred samples with % change within ±10% of the initial analysis results (Supplementary Fig. S2). 4. Conclusion A highly sensitive, selective and rapid UPLC–MS/MS method was developed for reliable measurement of 21-OH DFZ in human plasma. The current method is superior to all other methods with respect to sensitivity, selectivity, ruggedness and faster rate of analysis. The method can be readily used in a clinical setting where large number of samples is to be analyzed. The absence of matrix effect is effectively shown by post-column infusion and by the precision (% CV) values for the calculated slopes of calibration curves from different plasma sources. Additionally, incurred sample reanalysis of 115 samples proves the reproducibility of the validated method. Acknowledgements The authors are thankful to the management of Alkem Laboratories Ltd., Mumbai, India for providing instrumentation and infrastructure facility to carry out this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2013.07.035.

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