A Selective and Sensitive LC-MS/MS Method for the Determination of Beclomethasone Dipropionate and Beclomethasone-17-Propionate in Human Plasma

Xinping Fang, Dawei Zhou, Wenzhong Liang, and Zhe-ming Gu

XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, NJ 08536
Overview
A sensitive and specific liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method capable of quantifying beclomethasone dipropionate (BDP) and a metabolite, beclomethasone 17-propionate (B-17-P), in human plasma is described. The analytes were extracted from 0.5_mL plasma by a reverse phase (C18) solid phase extract method. The separation was achieved on a reverse phase (C18) HPLC column, and analytes, along with their respective internal standards, were monitored using an AB SCIEX API-5000 tandem mass spectrometer, employing turbo-ion spray ionization in the positive ion mode along with multiple reaction monitoring (MRM). The lower limit of quantitation (LLOQ) attained for BDP and B-17-P were 10 and 20 pg/mL, respectively. The method has been successfully applied to a concentration assay of human plasma samples as part of bioequivalence studies.
O
BDP

Structures
O O HO H H Cl H O O
Intensity, cps 500

Results and Discussion

Table I.
(A)
400 1.7e6 115.1 1.3e6 291.3 220 9.0e5 57.1 120 5.0e5 20 0.5 1.5 Time, min 2.5
3.01

Validation Data Summary of BDP in Human Plasma

10 to 2,500 pg/mL 0.991 to 0.998 Accuracy QC Conc. (pg/mL) 10 30 1000 2000 RE% -0.70 -5.33 -7.20 -6.50 Compared with Nominal Value (%) 62.40 Accuracy Condition RE% -10.5 ~ -1.33 -8.50 ~ -5.33 -8.50 ~ -4.67 -6.50 ~ 11.33 LLOQ Low Medium Precision CV% 12.9 9.23 2.88 4.30

301.3 279.3

319.3

(A)
337.2

Calibration Range 2 3 batches ) Correlation coefficient(r Accuracy & Precision Inter-Batch (n=15)

O
D D DD O O O O H O D D D D D

320

D HO H

147.2

185.0

277.3 263.2

393.3 373.3 355.3 429.2 503.2

Method Recovery

High

3.5

4.5

O O HO H H Cl O
B-17-P

Cl

H
BDP-D10

740 640

(B)

1.0e5 60

107.3

145.1 120

143.1

193.1

244.9

317.3

345.2

335.2

411.3
401.1 447.2 485.1 521.4

Freeze/Thaw Bench-To Autosampler Extract Stability Long-Term Storage Stability

3 Cycles,

-70 C

4 hrs, Room Temperature 3 Days, Room Temperature 61 Days, ~ -70 C

180

240

300 m/z, amu 360

420

480

540

Table II. Validation Data Summary of B-17-P in Human Plasma

1.20e6 301.3

OH H D D DD O

540 440 1.00e6 Intensity, cps

(B)
337.5

Calibration Range 2 3 batches ) Correlation coefficient(r Accuracy & Precision Inter-Batch (n=15) QC Conc. (pg/mL) LLOQ 20 Low 60 Medium 2000 High 4000

D HO H H Cl O H

340

OH H

240
2.90

8.00e5 6.00e5 4.00e5

120.0

140 40 0.5 1.5 Time, min 2.5 3.5 4.5

398.7 291.7 279.5 319.2 147.5 263.1 250 355.3 374.3 350 m/z, amu 450 550 434.2 416.1
Freeze/Thaw Bench-To Autosampler Extract Stability Long-Term Storage Stability

Introduction
Beclomethasone dipropionate (BDP) is a topically active corticosteroid used in the treatment of asthma and rhinitis. In recent years, several bioanalytical methods have been developed to support pharmacokinetic (PK) analysis in clinical trials, with the most sensitive method having a lower limit of quantification (LLOQ) of 50 pg/mL for both BDP and B-17-P in human plasma. BDP is extensively metabolized in the liver after oral administration, resulting in very low plasma levels. To improve PK evaluation in clinical trials, we have developed an LC-MS/MS method with increased sensitivity compared with existing methods. The LLOQs were lowered to 10 and 20 pg/mL for BDP and B-17-P, respectively.

513.6

Method Recovery

20 to 5,000 pg/mL 0.9970 to 0.9971 Accuracy RE% 4.00 1.83 -6.00 -4.50 Compared with Nominal Value (%) 62.56 Condition 3 Cycles, -70 C 4 hrs, Room Temperature 4 Days, Room Temperature 61 Days, ~ -70 C

Precision CV% 13.6 7.89 7.23 3.32

2.00e5 62.0

80.3

135.4 150

249.5

B-17-P-D5

Figure 1. Ion chromatograms of blank plasma (A), and 10 pg/mL BDP extracted from plasma (B)

50

Accuracy RE% -7.25 ~ -8.23 -5.75 ~ -5.67 -5.75 ~ 3.50 -0.25 ~ -2.17

LC-MS/MS Conditions
Liquid Chromatography: HPLC System Shimadzu LC-10AD Analytical Column: C18 column, 2.0 x 30 mm, 2.5 m Mobile Phase A) 0.4% acetic acid in water Mobile Phase B) Acetonitrile Mobile Phase C) Methanol (for column back wash) Gradient Flow rate: 0.5 mL/min Column Temperature: 50 C Injection Volume: 25 L
600 520 440 Intensity, cps 340

Figure 3. Product ion scan of BDP at 521.3 Da (A), and BDP-D10 at 531.3 Da (B).

(A)

2.2e6 1.8e6 1.4e6

279.3

(A)

In previous methods, two factors affected the detection limit: low extraction recovery and high matrix suppression. In addition, larger sample volumes (1-2 mL) were needed in order to achieve the desired LLOQ. Such conditions would require the use of individual extraction cartridges, which not only make sample preparation much longer, but could also introduce more error in the sample preparation procedure. A unique SPE method eluting with ethyl acetate and hexane dramatically increases the extraction recovery as well as decreases matrix interference, and the required LLOQs were achieved using only 0.5 mL of human plasma. Thus, sample clean-up can be done using a 96-well SPE plate, which makes sample preparation automation possible. Excellent linearity was obtained with a coefficient of determination (R2) greater than 0.991 for BDP and 0.997 for B-17P. The inter-day (n=15) precision (CV%) and accuracy (RE%) for all QC plasma samples, including LLOQ were 2.8812.9% and -7.20 -0.74%, respectively. (Table I) for BDP, and 3.3213.6% and -6.00 4.00%, respectively (Table II) for B-17-P. Three freeze/thaw cycles, 4-h ambient temperature storage, and 2 month long-term storage at -70 C QC appeared to have little effect on the quantitation.

240 140 40 1.3 600 500 400 1.6 1.9 Time, min 2.2 2.5 2.8 3.0 2.0e5 1.0e6 147.2 6.0e5 223.2 121.1 60 3.1e5 2.6e5 2.1e5 1.6e5 61.9 1.1e5 133.0 2.9 6.0e4 1.0e4 187.2 211.1 277.2 263.5 452.5 337.3 373.2 50 150 250 291.3 120 180 240 m/z, amu300 279.6 360 420 480 171.0 277.4 319.2 337.2 373.3 447.2

(B)
2.10

Experimental
Sample Preparation BDP, B-17-P, and the internal standards (BDP-D10 and B-17-P-D5) were transferred onto the preconditioned Strata C18-E 96-well plate (Phenomenex, precondition with 1 mL of methanol and 1 mL of water). The SPE plate was washed with 1.6 mL of water, 0.8 mL of 20% methanol in water, and 0.8 mL of Hexane. Analytes and internal standards were then eluted using a mixed solution of ethyl acetate and hexane. Mass Spectrometry MS System: Condition: MRM Transition: BDP: B-17-P: BDP-D10: B-17-P-D5:
Intensity, cps

(B)
135.5

300 200 100

1.27 3.02 1.96 2.01 1.31 1.38 1.13 1.23 1.43 1.51 1.61 1.71 1.74 1.88 2.19 2.41 2.49 2.24 2.33 2.61 2.85 2.68 2.79 2.90 2.97

521.3 465.2 531.3 470.2

301.2 279.3 301.2 279.2

1.1

1.4

1.7 Time, min2.0

2.3

2.6

Intensity, cps

AB Sciex API-4000 LC/(+)ESI-MS/MS (MRM)

Figure 2. Ion chromatograms of blank plasma (A), and 20 pg/mL B17-P extracted from plasma (B)

m/z, amu

350

450

550

Figure 4. Product ion scan of B-17-P at 465.2 Da (A), and B-17-P-D5 at 470.2 Da (B).