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Y. Wang, C.-Z. Wang, C. Yuan, Y. Chen, Z. Tan, W. Huang and H. Zhou, Anal. Methods, 2016, DOI:
10.1039/C6AY00202A.

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 Page 1 of 44 Analytical Methods
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DOI: 10.1039/C6AY00202A

1
2
3
4 1 Quantitative determination of betamethasone sodium phosphate and
5
6 2 ，
betamethasone dipropionate together with their metabolites betamethasone，
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8
9 3 betamethasone 17-monopropionate and betamethasone 21-monopropionate in
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10
11 4 human plasma by UPLC-MS/MS and a bioequivalence study

Analytical Methods Accepted Manuscript

12
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15 5 Man-Yun Chena,b, Yong-Jun Tanga,b, Yi-Cheng Wanga,b, Chong-Zhi Wangc, Chun-Su
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17
6 Yuanc, Yao Chena,b, Zhi-Rong Tana,b**, Wei-Hua Huanga,b,c*, Hong-Hao Zhoua,b
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19
a
20 7 Department of Clinical Pharmacology, Xiangya Hospital, Central South University,
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8 Changsha 410008, China
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24
b
25 9 Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics,
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10 Central South University, Changsha 410078, China
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29
c
30 11 Tang Center for Herbal Medicine Research, The Pritzker School of Medicine,
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12 University of Chicago; 5841 South Maryland Avenue, MC 4028, Chicago, IL 60637,
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35 13 USA
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40 15
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54 * **
55 / Corresponding authors. Tel.: +86 731-8480 5380, fax: +86 731-8235 4476;
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57 E-mails: endeavor34852@aliyun.com (W.-H. Huang); tanzr@163.com (Z.-R. Tan).
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 Analytical Methods Page 2 of 44
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3
1 ABSTRACT
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2 The compound medicine of betamethasone sodium phosphate (BSP) and
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8 3 betamethasone dipropionate (BDP) is widely used for diverse glucocorticoid-sensitive
9
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10 4 acute and chronic diseases such as asthma, rheumatoid arthritis and systemic lupus
11

Analytical Methods Accepted Manuscript

12 5 erythematosus. It will be useful and beneficial to validate sensitive method for the
13
14 6 determination of BSP, BDP and their metabolites for their pharmacokinetic study.
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16 7 Hereby, an ultra-high pressure liquid chromatography-tandem mass spectrometry
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18 8 (UPLC-MS/MS) has been validated for the determination of BSP, BDP and their
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20 9 metabolites betamethasone (BOH), betamethasone 17-monodipropionate (B17P) and
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10 betamethasone 21-monodipropionate (B21P) in human plasma. Liquid-liquid
23 11 extraction with ether and n-hexane (v/v, 4:1) was used for sample preparation of BDP,
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25 12 BOH, B17P and B21P with beclomethasone dipropionate as internal standard (IS),
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27 13 while solid phase extraction was adopted for sample preparation of BSP using
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29 14 prednisolone as IS. The chromatographic separation was performed on a Hypurity C18
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31 15 column (150 mm×2.1 mm, 5 µm) for BOH, BDP, B21P and B17P, and a Luna C18 (2)
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33 16 column (150 mm×2.0 mm, 5 µm) for BSP. Electrospray ionization interfaced with
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35 17 positive multiple reaction monitoring (MRM) scan mode was used for mass
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37 18 spectrometric detection. The standard calibration curves were linear within the range
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19 of 2.525 × 10-9-403.9 × 10-9 mol·dm-3 for BSP, 0.125 × 10-9-55.81 × 10-9 mol·dm-3 for
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40 20 BDP, 0.278 × 10-9-74.95 × 10-9 mol·dm-3 for BOH, 0.098 × 10-9-4.688 × 10-9
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42 21 mol·dm-3 for B17P and 0.226 × 10-9-5.411 × 10-9 mol·dm-3 for B21P, respectively. The
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44 22 validated method was successfully applied to a bioequivalence study in 23 healthy
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46 23 subjects after they were injected with this compound medicine BSP and BDP.
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49 24 Keywords: betamethasone; betamethasone sodium phosphate; betamethasone
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51 25 dipropionate; UPLC-MS; human plasma
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54 26
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1
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4 1 1. Introduction
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7 2 Both Betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BDP)
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9
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10 3 are synthesized glucocorticoid, which were able to reduce the production of

11

Analytical Methods Accepted Manuscript

12 4 inflammatory mediator.1 The compound betamethasone intramuscular injection
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15 5 consists of BSP and BDP, which were usually used for diverse
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6 glucocorticoid-sensitive acute and chronic diseases such as asthma, rheumatoid
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20 7 arthritis, systemic lupus erythematosus.2, 3
BSP and BDP could be hydrolyzed by
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8 phosphatase as fast-release phosphate prodrug and esterase enzymes as
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25 9 sustained-release dipropionate prodrug into active pharmaceutical ingredients
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10 betamethasone (BOH), betamethasone 17-monodipropionate (B17P) and
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30 11 betamethasone 21-monodipropionate (B21P), respectively.4, 5 Because the activating
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12 enzymes such as phosphate and esterase were highly efficient and ubiquitous in
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35 13 human blood, BSP that was highly ionized and hydrophilic could be rapidly absorbed
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14 into blood from administration place and then be metabolized quickly into active
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40 15 betamethasone (BOH) without rate-limiting step.6, 7 However, BDP was so highly
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43 16 lipophilic that it was slowly to release into the fluids of the intercellular space from
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45 17 muscle fibers, which resulted in the lower plasma concentration, longer half-life and
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48 18 slower metabolism process than BOH, B17P and B21P after intramuscular injection.6
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50 19 Therefore, it was useful and important to determine BSP, BDP and their metabolites in
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52
53 20 human plasma by a sensitive method.
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56 21 As synthesized glucocorticoid, the adverse effects are inevitable ranging from skin
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1
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4 1 fragility to full-blown iatrogenic Cushing syndrome such as obesity, hypertension,
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6 2 osteoporosis and other diseases.8, 9
Prolonged use of BSP and BDP may lead to
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9 3 suppression of the hypothalamic-pituitary-adrenal axis due to the impact on
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10
11 4 endogenous cortisol concentration as well as a circadian pattern.8, 10 Moreover, as a

Analytical Methods Accepted Manuscript

12
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14 5 kind of antenatal steroid therapy to prevent respiratory distress syndrome in premature
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16 6 neonates, it had been reported that maternal betamethasone administration could
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19 7 cause the risk of fetal growth restriction and preterm birth.11, 12 Hereby, developing
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21 8 and validating a sensitive analytical method for the quantification of BSP and BDP as
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24 9 well as their metabolites will be useful and valuable for their pharmacokinetic studies.
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26
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10 In the past three decades, there were several analysis methods validated to quantify
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30 11 BDP, BOH, B17P and B21P in human plasma, and the reported methods compared
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12 with this developed method will be discussed below. As mentioned above, BSP was
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35 13 rapidly and completely converted to BOH in vivo so that BSP was rarely determined
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14 for the quantification in human plasma in vitro and in vivo.13-15 In most studies, the
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40 15 pharmacokinetic processes of BSP and BDP were reflected by their main metabolites
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43 16 BOH and B17P due to the low blood concentration and poor receptor-binding affinity
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45 17 at the glucocorticoid receptor of B21P compared with B17P.16-18 In this study, the
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48 18 analytical method was accordingly validated to quantify BSP and BDP as well as their
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50 19 metabolites BOH, B17P and B21P in human plasma for a bioequivalence study.
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52
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54 20 As on-going development as the analytical instruments, ultra-performance liquid
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56 21 chromatography (UPLC) system had evolved from high performance liquid
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1
2
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4 1 chromatography (HPLC), which usually had packing material with smaller particle
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6 2 size less than 2 µm. Since UPLC has greatly improved the column efficiency and
7
8
9 3 resolution, it could shorten the analysis time.19 Especially equipped with mass
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10
11 4 spectrometer (MS), the sensitivity of UPLC-MS/MS was 3 to 5 times higher than

Analytical Methods Accepted Manuscript

12
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14 5 HPLC-MS/MS.20 Therefore, in this study, a rapid, simple and sensitive
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16 6 UPLC-MS/MS had been validated for the determination of BSP, BDP and their
17
18
19 7 metabolites BOH, B17P and B21P in human plasma. Then, the validated method was
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21 8 successfully applied to their pharmacokinetic studies in 23 healthy subjects after they
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24 9 were injected with this compound medicine BSP and BDP.
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26
27
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10 2. Experimental
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31 11 2.1. Chemicals and reagents
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33
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35 12 Standards of betamethasone sodium phosphate (BSP, purity: 99.4%, batch no.
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37 13 20120501), betamethasone dipropionate (BDP, purity: 99.7%, batch no. 120222),
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40 14 betamethasone (BOH, purity: 99.5%, batch no. 20101001), betamethasone
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42 15 17-monopropionate (B17P, purity: 98.1%, batch no. 20100601) and betamethasone
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44
45 16 21-monopropionate (B21P, purity: 99.3%, batch no. 20100602) were provided by
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47 17 Chongqing Huapont Pharm. Co., Ltd (Chongqing, China). The internal standard
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49
50 18 prednisolone (IS-1, purity: 99.6%, batch no. 100153-199603) and beclomethasone
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52 19 dipropionate (IS-2, purity: 99.9%, batch no. 100119-200603) were obtained from
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55 20 National Institute for Food and Drug Control (Beijing, China). Sodium cacodylate
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57 21 (purity: 99.5%, batch no.C1308011) was purchased from Aladdin Industrial
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4 1 Corporation (Shanghai, China). Potassium fluoride (purity: 99.2%, batch no.
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6 2 20120331) was bought from Chengdu Kelong Chemical Co. Ltd (Chengdu,
7
8
9 3 China). The structures of all the analytes and ISs were shown in Fig.1.The test
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11 4 drug (batch No.: 2012004) was supplied by the Chongqing Huapont Pharm. Co.,

Analytical Methods Accepted Manuscript

12
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14 5 Ltd, and the reference drug (batch No.: 2BBKA18A01) was purchased from
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16 6 Schering Plough pharmaceutical Co. Ltd (Shanghai, China).
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18
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20 7 Methanol (HPLC grade, batch no. I638607219) was purchased from Merck
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8 Company (Darmstadt, Germany). Deionized water was purified by a Millipore
23
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25 9 Milli Q-Plus system (Millipore, Bedford, MA, USA). The centrifuge (Type:
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10 Biofuge prime R) was produced by Heraeus company (Osterode, Germany).
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30 11 Formic acid (HPLC grade, batch no. HX090818), ammonium formate (HPLC
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12 grade, batch no. SX68594) and ammonium acetate (HPLC grade, batch no.
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35 13 SC12R2504) were supplied by CNW technologies GmbH (D ü sseldorf,
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37
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14 Germany). Ether (analytical grade, batch no. 20080311) and hexane (analytical
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40 15 grade, batch no. T20090225) were provided by Sinopharm Chemical Reagent
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43 16 Co., Ltd (Shanghai, China). Ammonia (analytical grade, batch no. 20090315)
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45 17 was purchased from Kaixin Chemical Reagent Co.,Ltd (Hunan, China). Blank
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48 18 human plasma was supplied by Changsha Blood Center (Changsha, China).
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50
51 19 2.2. Liquid chromatographic conditions
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53
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55 20 The Waters Acquity UPLC system (Acquity, Waters, USA) consisted of a
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57 21 vacuum degasser, a binary pump, an autosampler and a column compartment,
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1
2
3
4 1 which was used for chromatographic separation of samples. An API4000 mass
5
6 2 spectrometer produced by AB Sciex (Concord, Ontario, Canada) was coupled
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8
9 3 with the UPLC system for the analytes detection. The Analyte version 1.4.2
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10
11 4 software (Concord, Ontario, Canada) was used for collecting and analyzing data.

Analytical Methods Accepted Manuscript

12
13
14 5 The Hypurity C 18 column (150 mm × 2.1 mm, 5 µm) supplied by Thermo Fisher
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16 6 Scientific Inc. (Waltham, MA, USA) was installed for the separation of BOH,
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19 7 BDP, B21P and B17P with the maintenance temperature at 40 °C, while the
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21 8 Luna C 18 (2) column (150 mm × 2.0 mm, 5µm) purchased from Phenomenex Inc.
22
23
24 9 (Torrence, California, USA) was used for the isolation of BSP with the
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26 10 maintenance temperature at 40 °C. The C 18 columns of Security Guard
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29 11 Cartridges (4.0 mm × 3.0 mm, 5 µm, Part no.: AJ0-4378) purchased from
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31 12 Phenomenex Inc. (Torrence, California, USA) was assembled as the guard
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34 13 columns. The mobile phase used for all the analytes and the run-time were
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36 14 shown in Table 1, the flow rate of which was set at 0.3 mL/min. The
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38
39 15 temperature of the auto-sampler was maintained at 15 °C. 10 µL of the samples
40
41
16 was injected into the UPLC-MS/MS system.
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43
44
45 17 2.3. Mass spectrometric conditions
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47
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49 18 The effluent from the UPLC system was directed into the ESI probe. The mass
50
51 19 spectrometer was operated under the positive mode, the operating parameters of
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53
54 20 which were optimized with multiple reactions monitoring (MRM) detection to
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56 21 achieve maximal sensitivity by syringe infusion of working solutions
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1
2
3
4 1 containing each compound by peristaltic pump. Herein, the appropriate mass
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6 2 spectrometric signal responses of the precursor and product ions were calculated for
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8
9 3 identification and quantification of those compounds. In this study, the protonated
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11 4 precursor ions of BSP, BDP, BOH and B21P were chosen, while the precursor ion

Analytical Methods Accepted Manuscript

12
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14 5 [M+Na]+ was used for B17P due to its stronger response. The different and specific
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16 6 MS/MS parameters optimized for the developed method were presented in
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19 7 Table 2.
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8 2.4. Standard and quality control sample preparation
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24
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26 9 All stock solutions were prepared with methanol at the concentrations as following,
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29 10 2.997 × 10-3 mol·dm-3 for BOH, 2.344 × 10-3 mol·dm-3 for B17P, 2.705 × 10-3
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31 11 mol·dm-3 for B21P, 2.232 × 10-3 mol·dm-3 for BDP, 2.109 × 10-3 mol·dm-3 for BSP,
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34 12 2.871 × 10-3 mol·dm-3 for beclomethasone dipropionate (IS-1) and 2.750 × 10-3
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36 13 mol·dm-3 for prednisolone (IS-2), which were all stored at 4 °C until use.
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40 14 All working solutions were generated by diluting the stock solutions of all compounds
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42 15 step by step with the binary mixtures of methanol and 10mM ammonium formate (1:1,
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45 16 v/v). The upper limits of all analytes concentrations in the plasma samples used for
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47 17 standard calibration curves were prepared by spiking the drug-free human plasma
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50 18 with appropriate final working solutions. And, the quality control (QC) working
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52 19 solutions were prepared with the same procedures (Table 3). In order to obtain the
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55 20 final concentration of calibration curves, 50 µL working solution of each compound
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57 21 was spiked with 450 µL human blank plasma to make the desired concentration.
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1
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4 1 Because BSP is labile in human blank plasma, the resulting plasma solutions of BSP
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6 2 were mixed with human blank plasma, 2M sodium cacodylate and potassium fluoride
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9 3 (50%) (40:1:1, v/v/v) in advance (Table 3). The Beclomethasone dipropionate (IS-1)
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11 4 stock solution was further diluted with the binary mixtures of methanol and 10 mM

Analytical Methods Accepted Manuscript

12
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14 5 ammonium formate (1:1, v/v) as well to obtain the working IS solution at
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16 6 concentration of 287.1 × 10-9 mol·dm-3 used for BOH and 28.71 × 10-9 mol·dm-3 used
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19 7 for B17P, B21P, and BDP. The prednisolone (IS) working solution was prepared at the
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21 8 concentration of 550.0 × 10-9 mol·dm-3 used for BSP.
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25 9 2.5. Sample preparation
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29 10 500 µL of the collected human plasma was added into a disposable Eppendorf tube,
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31 11 which was followed by an addition of 50 µL of IS working solution, and subsequently
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34 12 vortexed for 30s on a vortex mixer (IKA VIBR AX, Germany). Continuously, one
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36 13 step of liquid-liquid extraction (LLE) was employed to extract BDP, BOH, B17P,
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39 14 B21P and IS from the human plasma, while solid phase extraction (SPE) was adopted
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41 15 for BSP sample preparation. For LLE, 2 mL of Ether/n-hexane (4: 1, v/v) as extractant
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44 16 was added into each tube with vortex-mixing for 10 min. Then, the well-mixed
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46 17 samples were centrifuged at 4000 rpm for 10 min, and 1.5 mL of supernatant organic
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49 18 layer was transferred into another Eppendorf tube and dried by a slow stream of
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51 19 nitrogen in water bath at 40 °C. Following, the residue was re-dissolved with 150 µL
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54 20 of methanol/10 mM ammonium formate (1:1, v/v) for BDP, BOH, B21P and
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56 21 methanol/water (7:3, v/v) for B17P by vortexing approximate 3 min, which was
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2
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4 1 re-centrifuged at 13000 rpm for another 5min before analysis. At last, 10 µL of
5
6 2 supernatant was injected into UPLC system.
7
8
9
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10 3 To the sample preparation of BSP, 500 µL of plasma sample mixed with 50 µL of IS

11

Analytical Methods Accepted Manuscript

12 4 working solution (550.0 × 10-9 mol·dm-3) was loaded into an activated OASIS HLB
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15 5 cartridge. After eluted by 1 mL of water/formic acid (50:1, v/v ) and 0.5 mL of water,
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6 the HLB cartridge was subsequently eluted by 1 mL of water/formic acid (50:1, v/v ),
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20 7 0.5 mL of water and 1 mL methanol/ammonia (20:1, v/v) under centrifuging at 1500
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22
8 rpm for 1 min, respectively. 0.6 mL of methanol/ammonia eluent was transferred into
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25 9 another Eppendorf tube and dried by nitrogen under water bath at 40 °C. The residue
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10 was re-dissolved with100 µL of methanol/20 mM ammonium acetate (1:1, v/v). After
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30 11 vortexed for approximately 3 min and centrifuged at 13000 rpm for 5 min, 10 µL of
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12 supernatant was injected into the UPLC system.
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36 13 2.6. Method validation
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40 14 The analytical method was validated according to FDA guidelines,21 which were
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42 15 pertinent to selectivity, sensitivity, linearity, precision, matrix effect, extraction
43
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45 16 recovery and stability reported on our previous study.22 The peak area ratios generated
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47 17 from the analytes of QC samples to IS were interpolated into the calibration curves
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49
50 18 plotted on the same day to directly calculate the concentrations of all the analytes. The
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52 19 QC samples were used in the procedure of evaluating the precision, accuracy and
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55 20 stability in three consecutive days.
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4 1 2.6.1. Selectivity
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6
7 2 The selectivity was evaluated by comparing the chromatographic profiles of six blank
8
9
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10 3 plasma samples from six different healthy subjects with those of corresponding
11

Analytical Methods Accepted Manuscript

12 4 plasma samples spiked with the analytes and IS, and those of plasma samples from
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15 5 drug subjects who had been injected with the compound medicine BSP and BDP.
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17
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19 6 2.6.2. Linearity and lower limit of quantification
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21
22 7 The calibration standards of all the analytes at seven concentrations were determined
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24
25 8 in three independent runs to validate the linearity. To avoid the interferences in human
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27 9 plasma, blank plasma samples were also analyzed. The peak area ratios for these
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29
30 10 compounds and IS were calculated to construct the calibration curves by plotting the
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32 11 peak area ratios of these compounds to IS versus the concentrations (x) of these
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35 12 analytes with weighted least squares linear regression (1/x). The lower limit of
36
37 13 quantification (LLOQ) was defined as the lowest concentration, which usually was
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40 14 the lowest concentration of the calibration curve with an acceptable precision (relative
41
42 15 standard deviation, RSD) and accuracy (relative error, RE). The values of LLOQs
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44
45 16 were at least the signal-to-noise ratios (S/N) of about 10. Five replicates were
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47 17 analyzed with an acceptable precision (RSD, within 20%) and accuracy (RE, 80–
48
49
50 18 120%) in this study.
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52
53
54
19 2.6.3. Precision and accuracy
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56
57 20 In order to evaluate intra-day precision and accuracy, six parallel and repeated QC
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2
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4 1 samples on high, middle, low concentration levels of these analytes were determined
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6 2 to obtain the calculated concentration using a calibration curve that was prepared and
7
8
9 3 analyzed on the same batch and day. The inter-day precision and accuracy was
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11 4 demonstrated by evaluating QC samples in three consecutive days. Six parallel and

Analytical Methods Accepted Manuscript

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13
14 5 repeated QC samples on high, middle, low concentration levels of these analytes were
15
16 6 determined on each day. The accuracy would be calculated by using mean
17
18
19 7 concentration results of QC samples at each level versus the nominal concentration,
20
21 8 which must be within 85%-115% (80%-120% for LLOQ).
22
23
24
25 9 2.6.4. Extraction recovery and matrix effect
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27
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29 10 The extraction recoveries of these analytes were analyzed and operated for six times
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31 11 by using the QC samples at different concentration levels. Recoveries were calculated
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33
34 12 by comparing the analyte/IS peak area ratio (A1) determined from extracted plasma
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36 13 samples with those (A2) from these QC sample solutions at the same concentrations.
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38
39 14 The matrix effects were calculated by comparing the peak areas of samples spiked
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41 15 post-extraction (A2) with those of the working solutions containing equivalent
42
43
44 16 amounts of these compounds (A3). The ratio values (A2/A3) ×100% were regarded
45
46 17 to be the matrix effects. The extraction recovery and matrix effect of ISs were also
47
48
49 18 calculated and operated with the same procedure.
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51
52 19 2.6.5. Stability
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54
55
56 20 The stability of all the analytes in human plasma was performed by analyzing triple
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2
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4 1 human plasma samples at different concentrations of QC samples for the sample
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6 2 storage and processing procedures. The freeze-thaw stability was conducted on QC
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8
9 3 plasma samples at different concentration levels, which were frozen at -40 °C for 24 h
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10
11 4 and thawed at room temperature (25 °C). The freeze-thaw cycles were operated twice,

Analytical Methods Accepted Manuscript

12
13
14 5 and then the samples were determined accordingly. The short-term stability was
15
16 6 evaluated after the exposure of the selected QC samples for 6h at room temperature
17
18
19 7 during the period that exceeded the routine time of sample preparation and the
20
21 8 ready-to-inject samples. The long-term stability was assayed by processing QC
22
23
24 9 samples at different concentration levels stored at low temperature (-40 °C) for a
25
26 10 period of 40 days. The post-preparative stability was performed by analyzing QC
27
28
29 11 samples stored under auto-sampler condition (4 °C) for 12 h. The stock solution
30
31 12 stability at room temperature was evaluated by determination of concentration of all
32
33
34 13 analytes and IS stock solutions that were deposited at room temperature for 6 hours.
35
36
37
38
14 2.7. Application to pharmacokinetic study
39
40
41 15 According to the guidelines, there was no gender factors considered for the
42
43
44 16 pharmacokinetic study, twenty-four healthy male volunteers (Age: 22 ± 4.5 years;
45
46 17 Height: 172 ± 15.5 cm; Weight: 75.5 ± 15.5 kg.) were enrolled in accordance
47
48
49 18 with the clinical protocols in the study. All the healthy subjects signed the informed
50
51 19 consent form prior to assessing medical history, physical examination,
52
53
54 20 electrocardiogram and standard laboratory test results of blood cell, urinalysis and
55
56 21 biochemical profile. There was one drop-out in this clinical study.
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1
2
3
4 1 The method was applied to evaluate the pharmacokinetics of the compound medicine
5
6 2 in the healthy subjects. Twenty-four healthy male volunteers received a single
7
8
9 3 intramuscular injection dose of Compound Betamethasone Injection (BSP (2 mg) and
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10
11 4 BDP (5 mg)) in an open label, two-periods, single-dose, two-way randomized

Analytical Methods Accepted Manuscript

12
13
14 5 crossover design with a washout period of forty-three days. Forearm venous blood
15
16 6 samples were collected from each subject at pre-dose(0 h), 5 min, 10 min, 15 min, 30
17
18
19 7 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 8 h, 12 h, 16 h, 24 h, 48 h, 72 h, 120
20
21 8 h, 168 h, 264 h and 360 h post-dose. 6 mL blood samples were transferred to
22
23
24 9 heparinized tubes which contained 90 µL 2M sodium cacodylate for 0-4 h. And 5 mL
25
26 10 blood samples were transferred to heparinized tubes which contained 75 µL 2M
27
28
29 11 sodium cacodylate for 360 h. All of blood samples were mixed immediately and
30
31 12 placed on the ice. After centrifuged at 4000 rpm for 10 min (4 °C), about 2-3 mL
32
33
34 13 plasma fractions were separated into Eppendorf tubes which contained 50% (w/v)
35
36 14 sodium fluoride to stabilize BSP. Then, the collected plasma samples were stored at
37
38
39 15 −40 °C prior to analysis. The pharmacokinetic parameters were calculated with
40
41
16 non-compartment model, using the “Drug and statistics for windows” software (DAS),
42
43
44 17 version 3.2.2. All of the pharmacokinetic parameters were calculated by using 90%
45
46
18 geometric confidence interval of the ratios (T/R) from the ANOVA with least-squares
47
48
49 19 means of the ln-transformed parameter.
50
51
52
53 20 The established method was applied to analyze the plasma concentrations of the
54
55 21 analytes, and then their pharmacokinetic parameters were calculated respectively. The
56
57
58 22 maximum plasma concentration (Cmax) and their time of occurrence (Tmax) were
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1
2
3
4 1 directly recorded on the concentration-time curve. The elimination rate constant (ke)
5
6 2 was calculated by the log-linear regression of concentrations observed during the
7
8
9 3 terminal phase of elimination, and the elimination half-life (T1/2) was then calculated
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10
11 4 as 0.693/ke. The area under the plasma concentration–time curve (AUC0–t) from the

Analytical Methods Accepted Manuscript

12
13
14 5 start of the infusion to the time of the last determined concentration was calculated
15
16 6 using the linear trapezoidal rule. The area under the plasma concentration–time curve
17
18
19 7 to time infinity (AUC0–∞) was calculated as follows: AUC0–∞= AUC0–t + Ct/ke.
20
21
22
8 3. Results and discussion
23
24
25
26 9 3.1. Method development and optimization
27
28
29
30 10 3.1.1. Sample preparation
31
32
33 11 The sample preparation was critical and imperative for any bio-analysis method. A
34
35
36 12 good sample preparation procedure could benefit valid chromatographic separation,
37
38 13 reliable signal response, higher sensitivity and selectivity. Solid phase extraction
39
40
41 14 (SPE), liquid–liquid extraction (LLE) and protein precipitation (PP) are conventional
42
43
15 and typical sample preparation approaches to avoid interferences from plasma
44
45
46 16 samples. However, PP was limited by low-selectivity with the reason that the
47
48
17 supernatant contained un-precipitated plasma components,23 which maybe influence
49
50
51 18 the mass spectrometry signal response. Moreover, PP was harmful to lengthen the
52
53
19 lifetime of column for a large number of samples. Therefore, LLE was finally
54
55
56 20 processed for the plasma sample preparation of BDP, BOH, B17P and B21P due to the
57
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1
2
3
4 1 good lipophilic property of these analytes and the drawbacks of PP.
5
6
7 2 Compared with previously reported extractant such as methyl tert-butyl ether
8
9
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10 3 (MTBE),24 diethyl ether-hexamethylene,4 acetone–chloroform (5:1, v/v),9

11

Analytical Methods Accepted Manuscript

12 4 ether-cyclohexane(4:1, v/v)6 and methylene chloride,8 ether/n-hexane (4:1, v/v) was
13
14
15 5 chosen as an ideal solvent to extract the BDP, BOH, B17P and B21P with high
16
17
6 extraction efficiency and minimal matrix effects. But a non-negligible drawback of
18
19
20 7 LLE was that hydrophilic compounds could not be extracted by water-immiscible
21
22
8 solvent. Thus, SPE was more appropriate as the pre-treatment approach of the plasma
23
24
25 9 samples containing BSP, which was a kind of highly ionized sodium phosphate. In
26
27
10 order to prevent that BSP was hydrolyzed in vitro by phosphatase and plasma esterase
28
29
30 11 during the blood collection and plasma reparation or storage, 200 µL 2M sodium
31
32
33
12 cacodylate and 200 µL potassium fluoride (50%) were added into the blank plasma
34
35 13 samples as stabilizer.25
36
37
38
39 14 3.1.2. Optimization of the chromatographic condition
40
41
42 15 Comparing with the reported columns including C18 RP column26, 27 and C8 column,28
43
44
45 16 a Hypurity C18 column (150 mm × 2.1 mm, 5 µm) was used for the separation of BDP,
46
47 17 BOH, B17P and B21P, while a Luna C18 (2) column (150 mm×2.0 mm, 5µm) that
48
49
50 18 could tolerate a broad pH value range (1.5-10) was installed for the analysis of BSP.
51
52 19 The mobile phase was a major factor to affect the retention time, resolution and peak
53
54
55 20 shape of analytes and IS. It was reported that acetonitrile preferred to produce
56
57 21 abundant solvent adduct ions [M+H+CH3CN]+ which could be observed on a
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1
2
3
4 1 triple-quadrupole mass spectrometry,24 so methanol was optimized as the organic
5
6 2 solvent with appropriate mass response. The pH value of mobile phase was another
7
8
9 3 critical factor that not only influenced the retention behaviors on column but also
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10
11 4 affected the ionization efficiency of the interested compounds. Ammonium formate

Analytical Methods Accepted Manuscript

12
13
14 5 and ammonium acetate were used as the pH modifier to keep the acidity of mobile
15
16 6 phase, which would easily keep the analytes positively charged.
17
18
19
20 7 The chromatographic conditions were optimized according to both chromatographic
21
22
8 resolution and shorter retention time. Finally, a mobile phase consisting of methanol
23
24
25 9 and water (70:30, v/v) was used for isocratic elution and quantification of BDP and
26
27
10 B17P in human plasma as well as methanol and water (63:37, v/v) for BOH. Besides,
28
29
30 11 a mobile phase consisting of methanol and water with 20 mM ammonium acetate
31
32
33
12 (70:30, v/v) was optimized for BSP as well as methanol and water with 10 mM
34
35 13 ammonium acetate (70:30, v/v) for B21P. Other chromatographic conditions were
36
37
38
14 presented in Table 1.
39
40
41 15 3.1.3. Optimization of the LC-MS/MS conditions
42
43
44
45 16 Different ionization sources such as electrospray ionization (ESI),
46
47 17 atmospheric-pressure chemical ionization (APCI) and atmospheric-pressure
48
49
50 18 photoionization (APPI) possessed different ionization processing for different
51
52 19 targeting compounds. ESI, APCI and APPI were usually used for polar, moderately
53
54
55 20 polar and non-polar compounds, respectively.29 According to the structures of BSP,
56
57 21 BDP, BOH, B17P and B21P (Fig. 1), ESI was selected for its higher ionization
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1
2
3
4 1 efficiency. The positive mode was used for the mass detection. In the full-scan spectra
5
6 2 of precursor and product ions, the multiple reaction monitoring (MRM) scan mode
7
8
9 3 was used for the quantification of the analytes with the transitions at m/z 473.4→
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10
11 4 435.4 for BSP, m/z 505.5→411.3 for BDP, m/z 393.2→373.3 for BOH, m/z 471.5→

Analytical Methods Accepted Manuscript

12
13
14 5 397.2 for B17P, m/z 449.4→411.2 for B21P, m/z 521.6→503.4 for IS-1 and m/z 361.5
15
16 6 →343.2 for IS-2 (Fig. 2).
17
18
19
20 7 In the ionized molecules, different neutral small molecules were the fragments from
21
22
8 the analytes and IS, respectively, which provided different product ion fragments
23
24
25 9 monitored for quantitative analysis. The chemical fragmentation schemes were
26
27
10 described in Fig. 2. The collision energy was optimized to satisfy the sensitivity in the
28
29
30 11 MRM mode, and the optimal values were provided at different voltage in Table 2.
31
32
33
34 12 3.2. Method validation
35
36
37 13 3.2.1. Selectivity
38
39
40
41 14 The developed method showed a good selectivity by comparing the typical MRM
42
43
15 chromatograms of all the compounds. Fig.3 (A-H) provided the typical MRM
44
45
46 16 chromatograms of the blank plasma, the blank plasmas spiked with different analytes
47
48
17 at their LLOQ concentrations. The typical MRM chromatograms of plasma samples
49
50
51 18 from a healthy subject at different time after injection of BSP and BDP were shown in
52
53
19 Fig.4 (A-D). As indicated in the chromatograms, there was no endogenous substance
54
55
56 20 to interfere the analysis of all the compounds, which proved the chromatographic
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1
2
3
4 1 conditions suitable and specific to the target analytes. The retention time in the
5
6 2 chromatograms was approximately 4.88, 5.04, 2.86, 2.99 and 3.01 min for BSP, BDP,
7
8
9 3 BOH, B17P and B21P in human plasma, respectively.
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10
11

Analytical Methods Accepted Manuscript

12
4 3.2.2. LLOQ and linearity
13
14
15
16 5 The calibration curves were constructed daily, which showed good linearity in the test
17
18
19
6 range of all these analytes and the mean regression equations were calculated from
20
21 7 five different calibration curves for each analyte in this study (Table 4). The peak-area
22
23
24 8 ratio of the analyte to IS versus the nominal concentration exhibited a good linear
25
26 9 relationship within the calibration range, respectively. The ideal IS for LC-MS
27
28
29 10 quantitation studies are deuterated versions of their target compounds, because only
30
31 11 limited kinetic isotope effects these compounds represent the most accurate mimic of
32
33
34 12 the target compound, its properties, retention, and response during ionization and
35
36 13 detection inside a mass spectrometer. Generally, there are two types of ISs, which are
37
38
39 14 structural analog and stable isotope labeled ISs. Whenever possible, stable isotope
40
41 15 labeled ISs should be used because they are most effective. Therefore, the deuterated
42
43
44 16 compounds will be the best internal standard used for the quantification of the
45
46 17 analytes. In this study, the desirable performances of stable isotope labeled ISs are not
47
48
49 18 available for us and too expensive to synthesize so that the structural analogs were
50
51 19 used as ISs. The selected internal standard that has the similar chemical structure with
52
53
54 20 the analytes and would not affect the analysis of the target compounds was employed
55
56 21 for the quantitative determination.30, 31 The precision (RSD%) and accuracy of each
57
58
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1
2
3
4 1 calibration curve was around 1.32-14.7% and 92.4-109.4% for BSP, 0.25-20.4% and
5
6 2 88.0-106.1% for BDP, 0.46-12.0% and 97.7-102.8% for BOH, 0.04-13.8% and
7
8
9 3 92.9-111.4% for B17P as well as 0.96-7.49% and 87.1-109.4% for B21P, respectively.
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10
11 4 The correlation coefficients of all the calibration curves were all＞0.99, which

Analytical Methods Accepted Manuscript

12
13
14 5 satisfied the linear regression requirement of biological samples. Moreover, the
15
16 6 appropriate linearity showed higher responses between the concentrations and peak
17
18
19 7 areas of analytes and ISs within the test ranges. The test ranges of all the objectives in
20
21 8 the calibration curves were provided in Table 4.
22
23
24
25 9 The precision and accuracy of LLOQs of five investigated compounds were 14.3%
26
27
10 and 94.7% for BSP, 11.6% and 88.1% for BDP, 9.10% and 111.3% for BOH, 18.4%
28
29
30 11 and 97.7% for B17P, 17.0% and 81.2% for B21P, which were all within ±20% and
31
32
33
12 80-120%, respectively. As the LLOQs shown in Table 4, the method was sensitive for
34
35 13 quantification of trace BSP, BDP and their metabolites BOH, B17P and B21P in
36
37
38
14 human plasma.
39
40
41 15 3.2.3. Intra- and inter-day precision and accuracy
42
43
44
45 16 The intra- and inter-day precision (RSDs) and accuracy (RE) for all those analytes
46
47 17 were presented in Table 5. In this validation, the inter- and intra-day precision
48
49
50 18 variations were within 4.41-17.1% and 2.86-15.0%, while the inter- and intra-day
51
52 19 accuracy variations were within 88.2-108.7% and 86.1-114.0%, respectively. The
53
54
55 20 overall data was effective with the requirements that precision must be ≤15% (≤20%
56
57 21 for around LLOQ) and accuracy will be based on the mean concentration, which must
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1
2
3
4 1 be within ±15% (±20% for around LLOQ) of the nominal concentration. The
5
6 2 results suggested that the developed method was accurate for quantifying investigated
7
8
9 3 compounds.
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10
11

Analytical Methods Accepted Manuscript

12
4 3.2.4. Extraction recovery and matrix effect
13
14
15
16 5 In the process of sample preparation, the LLE and SPE had been chosen to extract and
17
18
19
6 clean up the biological samples. Because the analytical signals may be suppressed or
20
21 7 enhanced for the presence of co-eluting substances of matrix to alert the ionization of
22
23
24 8 droplets,32 which affected the sensitivity, accuracy and precision of the method, the
25
26 9 extraction recovery and matrix effect were significant and essential to validate the
27
28
29 10 developed methods. The extraction recovery and matrix effects were exhibited in the
30
31 11 Table 6. All the extraction recoveries values of all those compounds were within
32
33
34 12 64.5–113.8 %. All results showed good efficiency of extraction and no endogenous
35
36 13 co-elutes interfering the ionization of all those investigated compounds.
37
38
39
40 14 3.2.5. Stability
41
42
43
15 Table 7 summarized the freeze–thaw stability, short-term stability, long-term stability
44
45
46 16 and auto-sampler stability data of all those analytes. The results indicated that all
47
48
17 samples were not labile without any obvious degradation during the routine analysis
49
50
51 18 of biological samples. Table 7 presented the stock solution stability data of all those
52
53
19 analytes and ISs. The results indicated that all the working samples were reliable
54
55
56 20 under the routine operating conditions.
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3
4 1 3.3. Clinical applications
5
6
7 2 The validated method was successfully applied to the pharmacokinetic studies of the
8
9
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10 3 compound medicine BDP and BSP. A randomized, two periods and single-dose
11

Analytical Methods Accepted Manuscript

12 4 protocol was adopted. In the clinical trial study, twenty-four healthy male subjects
13
14
15 5 were recruited, but one subject dropped out during the studies due to suffering from
16
17
6 fainting at the administration of injection. The representative chromatograms of the
18
19
20 7 plasma samples were plotted in Fig. 4, which was collected at different time from a
21
22
8 subject after injecting administration with BSP and BDP sustain-release drug.
23
24
25
26 9 As a sustained release parent drug, the plasma concentration of BSP in human body
27
28
29 10 was so low that could not be detected in the healthy subjects’ plasma samples. Hereby,
30
31 11 the pharmacokinetic parameters of BSP were unable to be calculated for estimating
32
33
34 12 the bioequivalence of the two pharmaceutical preparations. Instead, the
35
36 13 pharmacokinetic data of BOH and B17P, the main active metabolite of BSP, could be
37
38
39 14 calculated to provide sufficient information to compare the bioequivalence of the test
40
41 15 and reference formulations. In this study, as an inactive metabolite of BDP, the plasma
42
43
44 16 concentration of B21P was slightly above the LLOQ (0.226 × 10-9 mol·dm-3) of B21P.
45
46 17 Moreover, the trace plasma concentration of B21P could only be detected at a few
47
48
49 18 blood points. Therefore, B21P was also not fixed to evaluate the bioequivalence of
50
51 19 these two injections.
52
53
54
55 20 The pharmacokinetic data of BDP, BOH and B17P were applied to evaluate the
56
57 21 bioequivalence of two intramuscular compound betamethasone injections.
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2
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4 1 Additionally, it has been reported that BDP had a shorter elimination half-life time
5
6 2 (0.43 h) than BOH and B17P.5 Moreover, the plasma concentration of BDP could not
7
8
9 3 be determined in 4 h after the administration of this compound medicine. Thus, the
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10
11 4 plasma samples collected from 0 h to 4 h after the healthy volunteers injected this

Analytical Methods Accepted Manuscript

12
13
14 5 drug were adopted to calculate the pharmacokinetic parameters of BDP. Due to the
15
16 6 reported elimination half-life time of BOH (9.6 h) and B17P (80.8 h),4 the collected
17
18
19 7 plasma samples from 0 h to 120 h and 0 h to 360 h were analyzed to calculate the
20
21 8 pharmacokinetic parameters of BOH and B17P, respectively.
22
23
24
25 9 The pharmacokinetic parameters of BDP, BOH and B17P with intramuscular injection
26
27
10 of the test and reference formulations were displayed in Table 8. It could be noted that
28
29
30 11 a few extreme variance in human pharmacokinetic data reached beyond +/- 50% for
31
32
33
12 some measurements. Actually, the pharmacokinetic data were calculated for the
34
35 13 selected target compounds from their measured human plasma concentrations. The
36
37
38
14 value variance beyond +/- 50% for some measurements appeared due to possibly
39
40 15 some extensive metabolizers and some poor metabolizers of the compound medicine,
41
42
43 16 betamethasone in the healthy volunteers. Additionally, the different gene types of the
44
45 17 healthy subjects, which is responsible for the metabolism of this drug, might result in
46
47
48 18 the significant difference of the pharmacokinetic data. Therefore, we will further
49
50 19 investigate the pharmacogenomics and pharmacogenetics of this drug in Chinese
51
52
53 20 people. The mean plasma concentration-time profiles of BDP, BOH and B17P were
54
55 21 depicted in Fig. 5. Using logarithmic conversion and variance analysis of AUC0→4
56
57
58 22 (BDP), AUC0→120 (BOH), AUC0→360 (B17P), AUC0→∞ and Cmax, the results showed
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1
2
3
4 1 that the pharmacokinetic parameters showed no significant differences (p > 0.05)
5
6 2 between the different compound medicine BSP and BDP formulations. Two
7
8
9 3 one-sided t-test of AUC0→4 (BDP), AUC0→120 (BOH), AUC0→360 (B17P), AUC0→∞
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10
11 4 and Cmax indicated that Thigh and Tlow were greater than unilateral T0.05. The AUC0→4

Analytical Methods Accepted Manuscript

12
13
14 5 (BDP), AUC0→120 (BOH), AUC0→360 (B17P), AUC0→∞ and Cmax of test formulation
15
16 6 did not exceed the range of parameters with defined statistical 90% confidence
17
18
19 7 intervals within the 80–125%. The point estimate values of AUC0→4 (BDP),
20
21 8 AUC0→120 (BOH), AUC0→360 (B17P), AUC0→∞ and Cmax were calculated. The number
22
23
24 9 of subjects with an extrapolated part of AUCinfinity of 20% was 0. The confidence
25
26 10 intervals for t-test of log AUC0→4 (BDP), log AUC0→120 (BOH), log AUC0→360
27
28
29 11 (B17P), log AUC0→∞ and log Cmax were evidenced to be equivalent with statistical 90%
30
31 12 confidence intervals within the 80–125% for test/reference ratios of AUC0−t, AUC0−∞
32
33
34 13 and Cmax according to the US Food and Drug Administration.
35
36
37
38
14 3.4. Comparison with reported methods
39
40
41 15 The major advantage of above-mentioned method had good sensitivity of all those
42
43
44 16 analytes (LLOQs: 2.525 × 10-9 mol·dm-3 for BSP, 0.125 × 10-9 mol·dm-3 for BDP,
45
46 17 0.278 × 10-9 mol·dm-3 for BOH, 0.098 × 10-9 mol·dm-3 for B17P and 0.226 × 10-9
47
48
49 18 mol·dm-3 for B21P), which were lower than the methods previously reported on their
50
51 19 LLOQs: 3.875 × 10-9 mol·dm-3 for BSP,13 0.6-1.6 × 10-9 mol·dm-3 for BDP,8 0.112 ×
52
53
54 20 10-9 mol·dm-3 for B17P and 0.816 × 10-9 mol·dm-3 for BOH,4 especially without any
55
56 21 LLOQ for B21P reported. In the past decades, most of the validated HPLC-MS/MS
57
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1
2
3
4 1 were used for the quantification of BDP, BOH and B17P in both drug formulations
5
6 2 and biological samples,4-6, 8, 9, 28
but few methods were developed for the
7
8
9 3 determination of BSP and B21P in human plasma. Although BSP was not detected in
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10
11 4 the collected human plasmas from the healthy subjects due to its rapid degradation in

Analytical Methods Accepted Manuscript

12
13
14 5 human body, the validated method might be useful for the determination of the
15
16 6 sustained-release preparations containing BSP developed in the future. Besides,
17
18
19 7 GC-MS was also used for the analysis of BDP, BOH and B17P, but the sample
20
21 8 preparation procedure was labor-consuming and adding steps.29, 33, 34 In this study, the
22
23
24 9 UPLC system shortened the retention time of all these analytes, which were all less
25
26 10 than 6 min. The shorter run time was prerequisite condition to allow high sample
27
28
29 11 throughput for therapeutic drug monitoring such as the compound betamethasone
30
31 12 intramuscular injection. Thus, the developed methods were useful and meaningful for
32
33
34 13 the determination of BSP, BDP, BOH, B17P and B21P in human plasma.
35
36
37
38
14 4. Conclusions
39
40
41 15 A sensitive and reliable UPLC-MS/MS had been validated to quantify BSP, BDP and
42
43
44 16 their metabolites BOH, B17P and B21P in human plasma. The developed method was
45
46 17 compliantly validated according to FDA guidelines. The validated method showed a
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48
49 18 good sensitivity for the quantification of the mentioned compounds in different
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51 19 individuals’ human plasma samples. This method has successfully been applied to
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54 20 analyzing samples in bioequivalence studies of different compound medicine BSP and
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56 21 BDP formulations, which were demonstrated to be equal.
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4 1 Competing interests
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6 2 All the authors declare that they have no conflict of interests.
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10 3 Acknowledgements
11

Analytical Methods Accepted Manuscript

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4 This work was supported by the National Natural Scientific Foundation of China (no.
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16 5 81001471 to Z.-R. Tan, 31400306 to W.-H. Huang), Hunan Provincial Natural
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6 Science Foundation of China (No.2015JJ3156 to W.-H. Huang), China Postdoctoral
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21 7 Science Foundation (No. 2015M570692) and the fundamental research funds for the
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24 8 central universities of Central South University (no. 1681-7608040003 to W.-H.
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26 9 Huang).
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30 10 Author Contributions
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32 11 R.-Z. T., W.-H. H. and H.-H. Z. designed this study. M.-Y. C., Y.-C. W. and Y.-J. T.
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35 12 performed the experiments. W.-H. H., R.-Z. T., Y. C., C.-Z. W. and C.-S. Y. analyzed
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37 13 the data and described the figures. M.-Y. C. and W.-H. H. wrote the manuscript. All
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40 14 authors have read and approved the final manuscript.
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4 1 References
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7 2 1. Y. K. Zhang, H. Yang, J. Y. Zhang, L. J. Song and Y. C. Fan, Int J Clin Pract,
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10 3 2014, 68, 633.

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12 4 2. E. Dauden, A. Bewley, J. Lambert, G. Girolomoni, F. Cambazard and K. Reich,
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11 4 Metab Dispos, 2005, 33, 1124.

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14 5 17. S. L. Fialho, R. C. Siqueira, R. Jorge and A. Silva-Cunha, J Drug Deliv Sci
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16 6 Tec, 2007, 17, 93.
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39 15 ation/Guidances/UCM070107.pdf.
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4 1 26. X. Matabosch, O. J. Pozo, N. Monfort, C. Pérez-Mañá, M. Farré, J. Segura
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6 2 and R. Ventura, Drug Test Anal, 2015, 7, 663.
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9 3 27. L. Wang, Y. Y. Yang, T. S. Chung and X. Q. Chen, J Pharm Biomed Anal,
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11 4 2002, 28, 629.

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14 5 28. T. T. Qu, R. Zhang, B. J. Wang, X. Y. Liu, G. Y. Yuan and R. C. Guo, Yao xue
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16 6 xue bao, 2008, 43, 402.
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19 7 29. R. Schiewek, M. Lorenz, R. Giese, K. Brockmann, T. Benter, S. Gäb and O. J.
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21 8 Schmitz, Anal Bioanal Chem, 2008, 392, 87.
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24 9 30. P. J. Taylor, Clin Biochem, 2005, 38, 328.
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26 10 31. A. Tan and K. Awaiye, Use of internal standards in LC-MS bioanalysis. In: W.
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29 11 K. Li, J. Zhang and F. L. S. Tse(eds). New Jersey: Handbook of LC-MS
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31 12 Bioanalysis: Best Practices, Experimental Protocols, and Regulations, 2013, p
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34 13 217-229.
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36 14 32. K. J. Bronsema, R. Bischoff, N. C. van de Merbel, J Chromatogr B Analyt
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39 15 Technol Biomed Life Sci, 2012, 893-894, 1-14.
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16 33. S. Pereira Ados, L. S. Oliveira, G. D. Mendes, J. J. Gabbai and G. De Nucci, J
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44 17 Chromatogr B Analyt Technol Biomed Life Sci, 2005, 828, 27.
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18 34. D. Courtheyn, J. Vercammen, M. Logghe, H. Seghers, K. De Wasch and H. De
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4 1 Figure legends
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6 2 Fig.1. Chemical structures of all the analytes and internal standards (IS).
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9 3 Fig.2. Positive ion mass scan spectra and fragmentation pathways of BSP (A),
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11 4 BDP (B), BOH (C), B17P (D), B21P (E), IS-1 (F) and IS-2 (G).

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14 5 Fig.3. The typical MRM chromatograms of a blank plasma (A), blank plasma
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16 6 spiked with IS-1 (28.71 × 10-9 mol·dm-3) (B) and IS-2 (550.0 × 10-9 mol·dm-3)
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19 7 (C), LLOQ of BSP (D, 2.525 × 10-9 mol·dm-3), BDP (E, 0.125 × 10-9
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21 8 mol·dm-3), BOH (F, 0.278 × 10-9 mol·dm-3), B17P (G, 0.098 × 10-9 mol·dm-3)
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24 9 and B21P (H, 0.226 × 10-9 mol·dm-3).
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26 10 Fig.4. The MRM chromatograms of human plasma samples from a healthy
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29 11 subject after injected with the compound medicine BSP and BDP, which
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31 12 were collected in 4 h (BOH (B); B17P (C)) and 12 h (BDP (A); B21P (D)),
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34 13 respectively.
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14 Fig.5. Mean plasma concentration-time profiles of BDP, BOH and B17P in
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39 15 human plasma after injecting with the compound medicine BSP and BDP
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16 of reference or test drug, each point and bar represented the mean ± SD (n
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4 1 Table 1. Liquid chromatographic conditions for all the analytes
5
6 Mobile phase Run-time R.T.a Chromatographic
7 Analyte
A B A/B(v/v) (min) (min) Column
8
9 H2O (20 mM The Luna C (2) column
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18
BSP CH3OH 70:30 8 4.95 (150 mm × 2.0 mm, 5 µm)
10 CH3COONH4)
11 The Hypurity C column
18
BDP CH3OH H2O 70:30 7 5.05 (150 mm × 2.1 mm,

Analytical Methods Accepted Manuscript

12 5 µm)
13 The Hypurity C column
18
BOH CH3OH H2O 63:37 15 2.82 (150 mm × 2.1 mm, 5 µm)
14
The Hypurity C column
18
15 B17P CH3OH H2O 70:30 7 2.96 (150 mm × 2.1 mm, 5 µm)
16
H2O (10 mM The Hypurity C column
17 B21P CH3OH 70:30 7 18
2.98 (150 mm × 2.1 mm,
HCOONH4) 5 µm)
18
19 a
20 2 R.T. = Retention Time (mean values)
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4 1 Table 2. The optimized mass spectrometric parameters of all the compoundsa
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6 Precursor Product CE DT CAG CUR GS1 GS2 SV TEM DP EP
Analyte
7 ion (m/z) ion (m/z) (V) (ms) (psi) (psi) (psi) (psi) (V) (°C) (V) (V)
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9 BSP 473.4 435.4 17 300 6 10 35 35 4500 350 60 10
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BDP 505.5 411.3 16 200 6 10 30 30 4500 350 105 10
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12 BOH 393.2 373.3 15 300 6 10 35 35 4500 450 80 10
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14 B17P 471.5 397.2 25 300 6 20 30 30 5000 550 45 10
15 B21P 449.2 411.3 15 300 6 20 30 30 5000 450 50 10
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17 IS-1 521.6 503.4 14 200 6 10 35 30 4500 400 93 10
18 IS-2 361.5 343.2 16 300 6 10 35 35 4500 360 50 10
19 a
20 2 CE: collision energy; DT: dwell time; CAG: collision gas; CUR: curtain gas; GS1:
21 3 the nebulizing gas of nitrogen; GS2: turbo spray gas; SV: ion spray voltage; DP:
22 4 declustering potential; EP: entrance potential.
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3 1 Table 3. The solution concentrations of samples for standard calibration curves
4 2 and quality controls (× 10-9 mol·dm-3)a
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6 BSP BDP BOH B17P B21P
7 NO.
8 WS SCCS WS SCCS WS SCCS WS SCCS WS SCCS
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S8 4039 403.9 558.1 55.81 749.5 74.95 46.88 4.688 53.99 5.411
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11 S7 2019 201.9 279.1 27.91 374.7 37.47 33.48 3.348 38.55 3.855

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13 S6 1010 504.8 139.5 13.95 187.4 18.74 23.44 2.344 26.99 2.699
14 S5 404.1 40.41 46.51 4.651 93.68 9.368 11.72 1.172 19.28 1.928
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16 S4 202.0 20.20 23.25 2.235 46.84 4.684 7.813 0.781 13.50 1.350
17 S3 101.0 10.10 7.751 0.751 23.42 2.342 3.906 0.391 8.998 0.900
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19 S2 50.50 5.051 2.584 0.258 11.71 1.171 1.953 0.195 4.499 0.450
20 S1 25.25 2.525 1.292 0.129 5.855 0.586 0.978 0.098 2.249 0.226
21
22 HQCS 3231 323.1 446.4 44.64 599.5 59.95 37.50 3.750 43.18 4.318
23 MQCS 403.9 40.39 46.51 4.651 99.92 9.992 11.72 1.172 19.28 1.929
24
25 LQCS 50.48 5.048 2.480 0.248 5.551 0.556 1.953 0.195 4.499 0.450
26 a
27 3 WS: working solution; SCCS: standard calibration curve solution; HQCS: high
28 4 quality control solution; MQCS: middle quality control solution; LQCS: low quality
29 5 control solution.
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3 1 Table 4. The calibration curves, linear ranges and LLOQs of BSP, BDP and its
4 2 metabolites BOH, B17P, B21P (× 10-9 mol·dm-3)
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7 Analyte Regression equation r2 Test range LLOQ
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Y = (0.02519 ± 0.00136)X + (0.01827 ±

10 BSP 0.9945 2.525-403.9 2.525
11 0.00738)

Analytical Methods Accepted Manuscript

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13 Y = (1.93253 ± 0.25396)X + (0.08710 ±
14 BDP 0.9982 0.125-55.81 0.125
15 0.07639)
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17 Y = (0.48979 ± 0.05516)X + (0.00791 ±
18 BOH 0.9980 0.278-74.95 0.278
19 0.00765)
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21 Y = (3.98058 ± 0.37633)X + (0.02678 ±
22 B17P 0.9934 0.098-4.688 0.098
23 0.04087)
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Y = (0.43080 ± 0.03460)X + (0.00938 ±
25
B21P 0.9960 0.226-5.411 0.226
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0.00558)
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7 1 Table 5. Precision and accuracy for the quantification of BSP, BDP and their metabolites BOH, B17P and B21P in human plasmaa
8
Intra-day (n=6) Inter-day (n=18)

Analytical Methods Accepted Manuscript

9
10 Conc. Added
(× 10-9 mol·dm-3) Mean conc. measured Accuracy Mean conc. measured Accuracy
11 (MCM, × 10-9 mol·dm-3) ± SD
Precision (RSD, %) (MCM, × 10-9 mol·dm-3) ± SD
Precision (RSD, %)
(RE, %) (RE, %)
12
5.048 4.578 ± 0.500 90.7 10.9 4.907 ± 0.674 97.2 13.7
13
14 BSP 40.39 38.72 ± 5.793 95.9 14.9 41.88 ± 5.521 104 13.2
15
323.1 320.0 ± 29.30 99.0 9.16 332.6 ± 28.12 103 8.45
16
17 0.248 0.260 ± 0.038 104 14.6 0.260 ± 0.044 104 17.2
18
19 BDP 4.651 4.986 ± 0.143 107 2.86 4.736 ± 0.542 102 11.4
20 44.64 42.46 ± 2.327 95.1 5.48 43.15 ± 3.746 96.7 8.68
21
22 0.556 0.584 ± 0.051 105 8.81 0.605 ± 0.071 109 12.0
23 BOH 106 6.09 108 5.17
9.992 10.60 ± 0.645 10.77 ± 0.556
24
25 59.95 54.44 ± 4.681 90.8 8.60 59.82 ± 5.888 99.8 9.84
26
0.195 0.223 ± 0.025 114 11.4 0.199 ± 0.031 102 16.3
27
28 B17P 1.172 1.158 ± 0.112 98.9 9.61 1.152 ± 0.103 98.3 8.84
29
30 3.750 3.230 ± 0.121 86.1 3.74 3.308 ± 0.183 88.2 5.54
31 0.450 0.468 ± 0.053 103.73 11.2 0.434 ± 0.053 96.7 12.4
32
33 B21P 1.929 1.871 ± 0.154 97.06 8.23 1.775 ± 0.129 92.1 7.31
34 94.52 3.47 92.1 4.41
4.318 4.082 ±0.143 3.976 ± 0.176
35
a
36 2 Conc.: concentration; SD: standard deviation; MCM: mean concentration measured; CA: concentration added; RE% = (MCM/CA)×100;
37 3 RSD%=SD/MCM×100.
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7 1 Table 6. Extraction recovery and matrix effect of BSP, BDP, BOH, B17P and B21P using LLE
8
Recovery (%) (n=6) Matrix effect (%) (n=6)

Analytical Methods Accepted Manuscript

9
10 Analyte
11 Low conc.a Middle conc. High conc. ISb Low conc. Middle conc. High conc. IS
12
13 BSP 86.2 ± 6.79 87.5 ± 3.87 83.4 ± 2.15 88.3 ± 2.55 113.9 ± 7.31 102.8 ± 1.92 104.1 ± 1.80 96.1 ± 1.68
14
15 BDP 96.1 ± 8.45 94.3 ± 7.51 93.8 ± 7.37 83.1 ± 11.7 102.4 ± 10.4 95.4 ± 3.08 90.3 ± 4.02 98.1 ± 10.3
16
17 BOH 73.8 ± 15.04 69.9 ± 5.13 64.5 ± 4.53 81.7 ± 9.93 93.4 ± 2.87 96.7 ± 3.87 93.5 ± 3.74 106.9 ± 4.27
18
19 B17P 95.4 ± 18.03 98.1 ± 6.82 89.6 ± 17.0 96.6 ± 14.7 92.3 ± 15.4 89.9 ± 9.88 85.2 ± 13.7 108.0 ± 18.0
20
B21P 98.9 ± 6.82 93.7 ± 9.53 91.7 ± 4.89 96.5 ± 9.81 110.4 ± 13.1 109.8 ± 8.01 110.9 ± 7.98 90.7 ± 10.9
21
22 2
a
Conc.: concentration. bIS: international standard (IS-1 and IS-2).
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7 1 Table 7. Stability results of all the analytes in human plasma at selected QC levels (n = 5, × 10-9 mol·dm-3)a
8
Freeze–thaw (four Long-term (40 days,

Analytical Methods Accepted Manuscript

9 Short-term (6 h, r.t.) The autosampler (r.t.)
10 cycles, -40 °C) -40 ◦C)
Analyte CA
11 Dev. Dev. Dev. Dev. Dev.
MCM ± SD MCM ± SD MCM ± SD MCM ± SD (24h) MCM ± SD (48h)
12 (%) (%) (%) (24h, %) (48h, %)
13 5.048 4.905 ± 0.357 -2.84 4.988 ± 0.250 -1.19 4.994 ± 0.560 -1.07 5.252 ± 0.696 4.03 5.585 ± 0.616 10.63
14 -2.67 -2.55 -1.06 1.08 9.88
BSP 40.39 39.30 ± 5.785 39.36 ± 5.767 39.94 ± 5.576 40.83 ± 5.209 44.38 ± 5.581
15
16 323.1 315.1 ± 4.665 -2.50 310.9 ± 14.43 -3.78 320.0 ± 10.35 -1.02 313.6 ± 35.39 -2.94 359.2 ± 22.46 11.19
17 0.248 0.276 ± 0.042 11.2 0.258 ± 0.048 4.0 0.240 ± 0.012 -3.2 0.220 ± 0.016 -11.2 0.222 ± 0.020 -10.4
18
BDP 4.651 4.811 ± 0.135 3.45 4.746 ± 0.155 2.04 4.734 ± 0.141 1.80 4.304 ± 0.123 -7.45 4.387 ± 0.103 -5.68
19
20 44.64 44.94 ± 1.177 0.68 44.42 ± 0.893 -0.51 46.43 ± 1.399 4.01 41.59 ± 1.016 -6.84 43.33 ± 1.131 -2.95
21 0.556 0.577 ± 0.038 3.67 0.610 ± 0.048 9.63 0.597 ± 0.064 7.34 0.584 ± 0.056 5.04 0.569 ± 0.087 2.29
22
BOH 9.991 10.48 ± 0.645 4.88 10.87 ± 0.633 8.82 10.57 ± 0.577 5.78 10.20 ± 0.505 -2.05 10.38 ± 0.513 3.89
23
24 59.95 63.29 ± 2.673 5.54 64.74 ± 5.074 8.02 52.55 ± 2.816 -12.3 53.47 ± 2.906 -10.82 63.95 ± 3.102 6.68
25 0.196 0.221 ± 0.335 12.5 0.214 ± 0.020 9.09 0.205 ± 0.027 4.54 0.208 ± 0.022 5.68 0.185 ± 0.025 -5.68
26
B17P 1.172 1.250 ± 0.112 6.62 1.196 ± 0.112 2.08 1.241 ± 0.129 5.91 1.266 ± 0.094 7.80 1.114 ± 0.129 -5.02
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28 3.750 3.832 ± 0.330 2.2 3.435 ± 0.353 -8.39 4.185 ± 0.616 10.5 3.295 ± 0.304 -12.14 3.574 ± 0.531 -4.7
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12 Cmax (× 10-9 mol·dm-3) 189.7 ± 71.67 192.9 ± 76.69 52.76 ± 11.03 54.82 ± 13.23 2.250 ± 1.034 2.145 ± 1.018
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22 F(%) 101.70% ± 36.10% 93.7% ± 16.3% 113.5% ± 40.6%
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G: IS-2 ( m/z: 361.5→m/z:343.2)
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Mean plasma concentration of B17P (× 10-9 mol·dm-3) Mean plasma concentration of BOH (× 10-9 mol·dm-3) Mean plasma concentration of BDP (× 10-9 mol·dm-3)

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