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Journal of Chromatography B, 951–952 (2014) 78–88

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom epage: www.elsev ier .com/ locate /chromb

etermination of six components of Andrographis paniculata extractnd one major metabolite of andrographolide in rat plasma by liquidhromatography–tandem mass spectrometry

ian Wang, Wenqing Yang, Guanglin Wang, Pingming Tang, Yang Sai ∗

epartment of Drug Metabolism and Pharmacokinetics, Hutchison Medipharma Ltd., Shanghai 201203, PR China

r t i c l e i n f o

rticle history:eceived 4 November 2013eceived in revised form 17 January 2014ccepted 20 January 2014vailable online 28 January 2014

eywords:ndrographis paniculataMPL-004hemical componentsC–MS/MSharmacokinetic study

a b s t r a c t

Andrographis paniculata (AP) has been widely used in Asian countries to treat many kinds of dis-eases for several decades. Hutchison Medipharma Ltd. developed an aqueous ethanol extract ofA. paniculata (APE) named as HMPL-004 to treat inflammatory bowel diseases. The representativechemical components of HMPL-004 include andrographolide (AND), neoandrographolide (NAND),14-deoxyandrographolide (DAND), 14-deoxy-11,12-didehydro-andrographolide (DDAND), apigenin-7-O-�-d-glucuronopyranoside (AODG) and chlorogenic acid (CLA). HM5013620 is the major circulatingmetabolite of AND. The purpose of this study was to develop a bioanalytical method to determine all sevencompounds in rat plasma using liquid chromatography coupled to electrospray tandem mass spectrom-etry (LC–MS/MS). The assay was fully validated according to FDA guidelines. The LC–MS/MS detectionwas operated in the negative mode, and the multiple reaction monitoring (MRM) mode was used forthe quantification. The analyte extraction was performed by protein precipitation with acetonitrile after

adding a small volume (2% of the total volume) of 10% formic acid into plasma to stabilize AND underbench-top condition (ice-bath). The linear ranges of the analytes were 8–2000 ng/mL for DDAND and4–2000 ng/mL for others. Validation results demonstrate that AND, NAND, DAND, DDAND, CLA, AODGand HM5013620 can be rapidly, accurately, precisely and robustly quantified in rat plasma. Furthermore,the method was successfully applied to characterize the pharmacokinetic profiles of all seven compoundsin Sprague-Dawley rats after a single oral administration of 750 mg of HMPL-004.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Traditional Chinese medicines (TCMs) are used throughout theorld but owing to their complex composition and lack of char-

cterization of active components, it is difficult for developers toerform quality control and ensure clinical efficacy. In addition, it islso difficult to characterize the pharmacokinetic properties of theany structure-diverse components when they are administered

imultaneously.Andrographis paniculata (AP) is a member of the plant family

canthaceae. It has been widely used in Asian countries includinghina, India, and Thailand to treat upper respiratory tract infec-ion, HIV infection, ulcerative colitis, hepatitis, rhinitis, sinusitis,

∗ Corresponding author at: Department of Drug Metabolism and Pharmacoki-etics, Hutchison Medipharma Ltd., Building 4, No. 720 Cai Lun Road, Zhangjiangi-Tech Park, Shanghai 201203, PR China. Tel.: +86 21 50790088;

ax: +86 21 50793595.E-mail addresses: Yangs@hmplglobal.com, saiyang2006@gmail.com (Y. Sai).

570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2014.01.028

bacillary dysentery, enteritis, leprosy and other inflammatory andinfectious diseases [1–4]. AP extract (APE) are available in Chinaas oral preparations or injections for therapeutic use. The ChinesePharmacopoeia recommends a daily dose of APE oral preparationsof 0.63–1.26 g with a dehydroandrographolide (DDAND) contentgreater than 3.8%.

The aqueous ethanol extract of AP leaves has been devel-oped by Hutchison Medipharma Ltd. (HMP) named as HMPL-004to treat inflammatory bowel diseases including ulcerative colitis(UC) and Crohn’s disease (CD). HMPL-004 includes many kinds ofactive constituents. Andrographolide (AND), neoandrographolide(NAND), 14-deoxyandrographolide (DAND), and 14-deoxy-11,12-didehydroandro-grapholide (DDAND) have already been shownto be the major diterpenoid components in AP [5]. In addi-tion to these compounds, apigenin-7-O-�-d-glucuronopyranoside(AODG) and chlorogenic acid (CLA) were identified to be the major

representative flavonoid and polyphenol components in HMPL-004, respectively. Among these six components, AND was themost important one, showing anti-inflammatory and antimicro-bial activity [6,7]. Its pharmacokinetics has also been investigated

J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88 79

ND, D

ef[saoiHAiOmmsso[i

aps

2

2

oNolpD8t(L

Fig. 1. Structure information of AND, NAND, DA

xtensively. The previous reports of AND metabolism were mainlyocused on the phase II conjugated metabolites found in excreta8–10]. A study of metabolites in post-dose human plasma sampleshowed that the major circulating metabolite of AND was gener-ted via hydrolysis (In house data, not shown here). The structuref this hydrolysis metabolite has been confirmed through chem-cally synthesizing the corresponding standard compound namedM5013620 by the Medicinal Chemistry group of HMP. BesidesND, DDAND also can be hydrolyzed to form a major circulat-

ng metabolite but it is not accessible through chemical synthesis.ur previous study also found that AND was unstable in biologicalatrices, such as blood and plasma, that over 20% of AND can beetabolized to form dehydrated metabolite or GSH adducts after

torage on ice-water for 1 h and over 50% of AND can be gone aftertorage at room temperature for 1 h. Although a number of studiesf AND have been reported, the stability of AND was not recognized11–14] and is likely to have resulted in an underestimation of ANDn biological samples.

The aim of this study is to develop a simple, sensitive, selectivend robust method to quantitatively determine all seven com-ounds in rat plasma and then apply it to in vivo pharmacokinetictudies.

. Materials and methods

.1. Chemicals and reagents

HMPL-004 was provided by the Pharmaceutical Science groupf HMP (Shanghai, China), containing 6.5% of AND, 3.1% ofAND, 3.4% of DDAND, 0.4% of DAND, 1.9% of CLA and 1.0%f AODG. AND (Purity 97.3%) was purchased from Guilin San-eng Biotech Co., Ltd. (Guangxi, China). NAND (Purity 93.4%) wasurchased from Zhongxin Innova Laboratories (Tianjin, China).AND (Purity 97.0%), DDAND (Purity 97.4%), and AODG (Purity

9.6%) were all purified and characterized from HMPL-004 byhe Pharmaceutical Science group of HMP (Shanghai, China). CLAPurity 96.4%) was purchased from Sichuan Weikeqi Biotech Co.,td. (Sichuan, China). HM5013620 (Purity 98.0%) was chemically

DAND, CLA, AODG, HM5013620 and cortisone.

synthesized by the Medicinal Chemistry group of HMP (Shanghai,China). Cortisone, as the internal standard (Purity 98.0%), was pur-chased from Sigma–Aldrich (St Louis, MO, USA). The structuresof all seven compounds and the internal standard are shown inFig. 1. Demethylsulfoxide (DMSO), formic acid and acetic acid wereall HPLC grade and purchased from Tedia (Fairfield, OH, USA).Methanol, acetonitrile, and isopropanol were all HPLC grade andpurchased from Thermo-Fisher Scientific (Pittsburgh, PA, USA).Deionized water (≥18.2 m�) was prepared by a Millipore Milli-Qgradient water purification system (Billerica, MA, USA).

2.2. Instruments

The analysis was performed on a triple quadrupole mass spec-trometer equipped with turbo ionspray interface (API4000, ABSciex, Foster City, CA, USA). An Agilent 1200 series liquid chro-matography system was used consisting of a binary pump, anon-line vacuum degasser and a thermostated column compartment(Agilent Technologies, Palo Alto, CA, USA) and a CTC PAL autosam-pler (CTC Analytics AG, Zwingen, Switzerland). Data acquisition andprocessing were performed using Analyst version 1.4.2 software(AB Sciex).

2.3. LC–MS/MS conditions

Analytes were separated on a Waters Xbridge Shield RP C18column (50 mm × 2.1 mm ID, particle size 5 �m; Waters Corp.,Mildford, MA, USA) protected with a Supelco filter (Particle size0.5 �m; Sigma–Aldrich, St Louis, MO, USA). The column temper-ature was maintained at 30◦C. Chromatography was performedusing a gradient system consisting of mobile phase solutionA (0.1% acetic acid in water) and solution B (50% acetoni-trile in methanol). The gradient elution method was as follows:0.00–2.00 min: 5% B→55% B; 2.00–3.70 min: 55% B; 3.70–3.71 min:

55% B→100% B; 3.71–5.00 min: 100% B; 5.00–5.01 min: 100% B→5%B; 5.01–6.50 min: 5% B. The flow rate was 0.4 mL/min and theautosampler temperature was set to 8◦C. During the first 0.60 minand after 5.10 min, the eluents were directed to waste. The injection

80 J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88

Table 1The MS parameters of each analyte.

Analyte Q1 (m/z) Q3 (m/z) DP EP CE CXP

AND 409 331 −40 −10 −18 −5NAND 539 59 −55 −10 −56 −9DAND 393 59 −35 −10 −35 −6DDAND 331 303 −80 −10 −34 −9CLA 353 191 −45 −10 −24 −3

vvbcm

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2

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2

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2

tpwn1s

AODG 445 269

HM5013620 367 305

IS (Cortisone) 359 329

olume was 10 �L. To avoid carryover, the syringe and injectionalve of the autosampler were set to be washed for 3 and 5 timesefore and after injection with wash solution 1 (90% isopropanolontaining 0.1% NH4OH) and wash solution 2 (0.1% acetic acid inethanol/acetonitrile/water (1:1:2, v/v/v)), respectively.The mass spectrometer was operated in the negative mode. The

onspray voltage and the source temperature were set to −4500 Vnd 500 ◦C, respectively. The nebulizer gas, heater gas, curtain gasnd collision gas were set to 60, 50, 20 and 6 psi, respectively. Theultiple reaction monitoring (MRM) mode was used for analyte

uantification, and the MS parameters of each analyte are listed inable 1.

.4. Preparation of stock and working solutions

For each of the seven compounds, two stock solutions1.0 mg/mL) were separately prepared in DMSO: one for the cal-bration standards and one for the quality control (QC) samples.n appropriate volume of stock solution of each compound for thealibration standards was spiked and mixed together firstly, theniluted in acetonitrile to obtain eight calibration standard work-

ng solutions containing AND, NAND, DAND, DDAND, CLA, AODGnd HM5013620, each at concentration levels of 40,000, 20,000,0,000, 4000, 1000, 400, 160 and 80 ng/mL. The QC working solu-ions containing the seven compounds were similarly prepared atoncentration levels of 32,000, 3000, 400, 160 and 80 ng/mL. For thenternal standard (IS), the stock solution was prepared at 1.0 mg/mLn acetonitrile and then diluted in acetonitrile to obtain the workingolution at 500 ng/mL. All solutions were stored at 4 ◦C.

.5. Preparation of calibration standard and QC samples

Calibration standards were prepared by adding small volumes5% of the total volume) of working solutions into blank heparinizedat plasma to give final concentrations of 2000, 1000, 500, 200, 50,0, 8 and 4 ng/mL for each analyte. QC samples were similarly pre-ared at concentration levels of 1600 (high QC), 150 (medium QC),0 (low QC for DDAND), 8 (lower limit of quantification (LLOQ)or DDAND and low QC for others) and 4 (LLOQ for all exceptDAND) ng/mL. A preliminary stability study showed that AND wasnstable in rat plasma, while formic acid can help to stabilize it. Forhis reason, the blank plasma was firstly acidified with a small vol-me (2% of the total volume) of 10% formic acid before the additionf working solution into blank plasma.

.6. Sample preparation

A protein precipitation method was used for sample prepara-ion. Simply, 150 �L of IS working solution was added into 50 �L of

lasma sample. After being vortexed for at least 1 min, the mixtureas centrifuged at 20,000 × g for 10 min at 4 ◦C. 100 �L of the super-atant was then transferred into another polythene tube containing00 �L of water, and vortexed well before injection into LC–MS/MSystem for analysis.

−60 −10 −36 −7−80 −10 −30 −9−78 −10 −14 −9

2.7. Validation of the method

The method was validated for specificity, selectivity, linearity,LLOQ, accuracy and precision, extraction recovery, matrix effect,hemolyzed plasma effect and stability according to US FDA guide-lines [15].

2.7.1. Specificity and selectivitySix different batches of analyte-free heparinized rat plasma

were used to investigate whether the endogenous matrix con-stituents interfered with the assay. The peak area at the retentiontime for each analyte should be no more than 20% of the peak areaof the LLOQ standards.

2.8. Linearity and LLOQ

The linearity of the method was derived from three separateanalytical runs. In each analytical run, calibration curves contain-ing double blank sample (no analyte or IS), blank sample (only IS)and eight (seven for DDAND) non-zero concentration levels wereprepared and analyzed in duplicate. Calibration curves in each ana-lytical run were fitted by the peak area ratio (Analyte/IS) versusanalyte concentrations using a 1/x2 weighted linear least-squaresregression model. LLOQ was defined as the lowest drug concen-tration on the calibration curve. The bias between the nominalconcentration and the back-calculated concentration should be lessthan 15%, except at LLOQ where the bias can be less than 20%.

2.9. Accuracy and precision

The intra-day accuracy and precision were evaluated at five(four for DDAND) concentration levels by the determination of sixreplicate QC samples on the same day. The inter-day accuracy andprecision were assessed at five (four for DDAND) concentration lev-els by analyzing six replicate QC samples over three consecutivedays. Precision was calculated as relative standard deviation (RSD),which should not exceed 20% for the LLOQ samples and 15% forother QC samples. The deviation of the mean values between thenominal concentration and the back-calculated concentration (RE)was used to evaluate the accuracy, which should be within ±20%for the LLOQ samples and ±15% for other QC samples.

2.9.1. Extraction recovery and matrix effectThe extraction recovery of each analyte was determined at three

concentration levels by comparing the mean peak areas of the reg-ularly pre-treated QC samples with those of the samples preparedby directly adding QC working solutions into the post-precipitatedblank plasma.

The matrix effect was also assessed at three concentration lev-els by comparing the mean peak areas of the samples prepared

by directly adding QC working solutions into the post-precipitatedblank plasma with those of the samples prepared by directlyadding QC working solution into neat final injected solution(acetonitrile/H2O = 1:1, v/v).

J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88 81

Fig. 2. Product ion scans and proposed fragmentation patterns of AND (A), NAND (B), DAND (C), DDAND (D), CLA (E), AODG (F), HM5013620 (G) and cortisone (H).

8 togr. B

2

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2 J. Wang et al. / J. Chroma

.10. Stability

The stability of each analyte was checked by analyzing threeeplicates of plasma samples at low QC and high QC, which werexposed to different storage conditions including bench-top stabil-ty (2.5 h on ice-water), three freeze/thaw cycle stability (−80 ◦C)nd long-term storage stability (−80 ◦C for 32 days). In addition, the

tability of each analyte was evaluated in plasma extracts exposedo the storage condition of autosampler (8 ◦C for 72 h). All analytesere considered stable when the deviation between the nominal

ig. 3. Representative LC–MS/MS chromatograms from blank rat plasma, spiked plasma scortisone).

951–952 (2014) 78–88

concentration and the mean value of the back-calculated concen-tration (RE) was within ±15%.

2.11. Hemolyzed plasma effect

The hemolyzed plasma was prepared by adding 10% (v/v) of

rat whole blood to rat plasma and vigorously vortexing to ensurethe blood hemolyzed and mixed well with the plasma. The effectsof hemolysis on AND, NAND, DAND, DDAND, CLA, AODG andHM5013620 were evaluated by spiking the working solution to

amples at the LLOQ of AND, NAND, DAND, DDAND, CLA, AODG, HM5013620 and IS

J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88 83

(Cont

htwtvc

2

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Fig. 3.

emolyzed plasma matrices with six replicates at three concen-ration levels. The spiked QC samples were extracted and analyzedith a set of regular plasma standards. The measured concentra-

ion values were compared with the nominal values. If the RSDalues did not exceed 15% and RE values were within ±15%, it wasonsidered that the effect of hemolysis could be neglected.

.12. Pharmacokinetic study

The validated LC–MS/MS method was applied to investigatehe pharmacokinetic profiles of each component of HMPL-004 in

inued ).

Sprague-Dawley (SD) rats (n = 6, male/female = 1:1) following asingle oral administration of 750 mg/kg of HMPL-004. The studyprotocol was evaluated and approved by Institutional AnimalCare and Use Committee (IACUC) of HMP. Venous blood samples(<0.15 mL) were collected in heparinized tubes containing 3 �L of10% formic acid at 0 (pre-dose), 15, 30 min and 1, 1.5, 2, 4, 6, 8, 12and 24 h post dosing from the retro-orbital sinus after the rat was

anesthetized by isoflurane. After inverting the tubes gently at least5 times, blood samples were centrifuged at 3000 × g for 10 min toseparate supernatant plasma, which was then stored at −80 ◦C untilanalysis.

84 J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88

Table 2The regression equations, linear range and LLOQ for the determination of all seven analytes.

Analyte Analytical run Regression equation Coefficient (R) Linear range (ng/mL) LLOQ (ng/mL)

AND1 y = 0.000857x + 0.000677 0.9977 4–2000 42 y = 0.000971x + 0.00072 0.99703 y = 0.00101x + 0.000277 0.9965

NAND1 y = 0.00117x + 0.000721 0.9974 4–2000 42 y = 0.00139x + 0.000973 0.99723 y = 0.00140x + 0.000353 0.9964

AODG1 y = 0.000925x + 0.000109 0.9989 4–2000 42 y = 0.00110x + 0.000118 0.99893 y = 0.00106x + 0.000796 0.9982

CLA1 y = 0.000923x + 0.000138 0.9983 4–2000 42 y = 0.000982x + 0.000518 0.99873 y = 0.000982x + 0.000712 0.9979

HM50136201 y = 0.000653x + 0.000254 0.9980 4–2000 42 y = 0.000790x + 0.000242 0.99813 y = 0.000732x + 8.91e005 0.9977

DAND1 y = 0.000375x + 0.000338 0.9968 4–2000 42 y = 0.000428x + 0.000307 0.99503 y = 0.000455x + 0.000149 0.9974

DDAND1 y = 9.14e−005x + 4.44e−005 0.9966 8–2000 82 y = 0.000101x + 0.00019 0.99793 y = 0.000106x + 9.51e006 0.9971

Table 3The intra-day and inter-day accuracies and precisions for the analysis of all analytes (n = 3 days, 6 replicates per day).

Analyte Nominal conc. (ng/mL) Intra-day Inter-day

Measured conc. (ng/mL) Accuracy (%) Precision (%) Measured conc. (ng/mL) Accuracy (%) Precision (%)

AND

4 3.58 ± 0.44 −10.46 12.37 3.81 ± 0.41 −4.65 10.818 7.56 ± 0.32 −5.54 4.29 7.72 ± 0.45 −3.45 5.83

20 19.00 ± 0.59 −5.00 3.12 19.50 ± 0.97 −2.50 4.95150 140.67 ± 5.61 −6.22 3.99 138.67 ± 4.56 −7.56 3.29

1600 1528.33 ± 45.35 −4.88 2.97 1505.56 ± 46.81 −5.90 3.11

NAND

4 3.56 ± 0.19 −11.08 5.20 3.70 ± 0.26 −7.43 7.138 7.60 ± 0.32 −5.04 4.18 7.63 ± 0.35 −4.69 4.60

20 18.63 ± 0.61 −6.83 3.25 19.05 ± 0.71 −4.75 3.71150 141.17 ± 6.94 −5.89 4.92 138.22 ± 5.87 −7.85 4.24

1600 1506.67 ± 39.33 −5.83 2.61 1479.44 ± 48.69 −7.53 3.29

DAND

4 3.45 ± 0.36 −13.88 10.38 3.89 ± 0.48 −2.76 12.258 7.34 ± 0.70 −8.27 9.58 7.84 ± 0.70 −1.98 8.89

20 18.23 ± 0.92 −8.83 5.02 19.34 ± 1.35 −3.28 7.00150 136.50 ± 8.53 −9.00 6.25 138.17 ± 7.60 −7.89 5.50

1600 1513.33 ± 69.47 −5.42 4.59 1476.11 ± 77.09 −7.74 5.22

DDAND

8 7.93 ± 0.64 −0.83 8.02 7.90 ± 0.85 −1.26 10.7920 20.25 ± 1.12 1.25 5.54 19.43 ± 1.37 −2.86 7.08

150 145.50 ± 5.96 −3.00 4.09 141.33 ± 5.83 −5.78 4.131600 1595.00 ± 61.24 −0.31 3.84 1551.67 ± 70.15 −3.02 4.52

CLA

4 3.79 ± 0.18 −5.29 4.85 3.83 ± 0.24 −4.31 6.328 7.74 ± 0.27 −3.23 3.43 7.70 ± 0.43 −3.81 5.58

20 18.40 ± 0.72 −8.00 3.89 18.46 ± 0.69 −7.69 3.74150 138.33 ± 8.07 −7.78 5.83 133.78 ± 8.71 −10.81 6.51

1600 1615.00 ± 161.59 0.94 10.01 1617.78 ± 131.17 1.11 8.11

AODG

4 3.94 ± 0.34 −1.58 8.75 3.97 ± 0.30 −0.76 7.468 7.92 ± 0.39 −1.02 4.98 7.72 ± 0.46 −3.55 5.95

20 18.48 ± 0.78 −7.58 4.20 18.58 ± 0.92 −7.08 4.95150 138.00 ± 5.62 −8.00 4.07 133.06 ± 7.94 −11.3 5.97

1600 1596.67 ± 111.12 −0.21 6.96 1552.22 ± 94.72 −2.99 6.10

HM5013620

4 3.54 ± 0.20 −11.63 5.79 3.71 ± 0.26 −7.32 7.138 7.87 ± 0.28 −1.67 3.54 7.67 ± 0.30 −4.10 3.96

20 18.87 ± 1.07 −5.67 5.66 18.96 ± 0.85 −5.19 4.46150 141.50 ± 4.14 −5.67 2.92 136.39 ± 7.37 −9.07 5.40

1600 1528.33 ± 84.00 −4.48 5.50 1500.56 ± 70.50 −6.22 4.70

J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88 85

Table 4Matrix effect and extraction recovery for the analytes in rat plasma (n = 6).

Analyte Nominal conc. (ng/mL) Extraction recovery Matrix effect

Mean SD RSD (%) Mean SD RSD (%)

AND8 119.86 6.38 5.33 85.66 2.18 2.55

150 103.62 3.21 3.10 90.43 3.08 3.401600 98.68 3.76 3.81 88.41 0.60 0.67

NAND8 119.69 6.20 5.18 89.35 3.76 4.21

150 105.01 2.19 2.09 91.40 3.82 4.181600 99.27 4.17 4.20 89.90 1.04 1.16

DAND8 114.91 12.81 11.15 93.60 7.56 8.07

150 103.75 4.16 4.01 95.32 3.04 3.191600 101.03 5.52 5.47 91.82 3.97 4.33

DDAND20 126.16 8.82 6.99 98.00 7.67 7.83

150 104.82 3.04 2.90 96.55 5.83 6.041600 103.23 5.78 5.60 93.62 2.75 2.94

CLA8 94.60 9.36 9.89 107.20 5.80 5.41

150 63.44 4.13 6.52 111.32 3.20 2.881600 63.11 1.25 1.98 112.13 1.01 0.90

AODG8 113.83 11.01 9.67 103.04 7.92 7.69

150 81.83 4.20 5.14 105.00 2.91 2.771600 78.98 2.42 3.07 104.83 1.14 1.09

8 112.60 6.52 5.79 96.92 3.30 3.40.91

.17

3

3

lf(tDmanaihawaaadtNs(cd3dcuiafws

HM5013620 150 94.12 11600 91.12 3

. Results and discussion

.1. Mass spectrometry

Q1 full scans were conducted by direct infusion of all the ana-ytes in a solution of acetonitrile-water (1:1, v/v) containing 0.1%ormic acid under positive and negative electrospray ionizationESI) modes. Better responses were observed in negative mode thanhose in positive mode for all compounds with the exception ofDAND which showed a little higher signal intensity in positiveode than that in negative mode. This phenomenon is predictable

s all the analytes are weakly acidic with DDAND the closest to aeutral compound. A number of studies have reported that aceticcid is an ideal negative-ion ESI modifier which can significantlyncrease the negative-ion ESI response of the analytes due to theigh gas-phase proton affinity of its anion, its small anion volumend its high acidity [16–18]. In this study, after 0.1% formic acidas replaced with 0.1% acetic acid, the negative ion responses of

ll analytes were increased significantly. Especially for AND, NANDnd DAND, the acetic acid adducts were formed so that the neg-tive signal intensities were enhanced more significantly. Fig. 2epicts the MS/MS product ion scans and proposed fragmenta-ion patterns of all analytes. Clear responses were shown for AND,AND and DAND at m/z of 409, 539 and 393 respectively, corre-

ponding to the deprotonated molecular ions of acetic acid adducts[M+HOAC–H]−), while for DDAND, CLA, AODG, HM5013620 andortisone, no acetic acid adducts were formed and only high abun-ant peaks of deprotonated molecular ions ([M−H]−) at m/z of 331,53, 445, 367 and 359 were found respectively. The most abun-ant product ions of AND, NAND and DAND were all at m/z of 59orresponding to the deprotonated ion of acetic acid and the prod-ct ions generated by loss of acetic acid also showed high signal

ntensities at m/z of 349, 479 and 333, respectively. For AND, an

bundant product ion at m/z of 331 was generated additionally byurther loss of water from m/z of 349. The molecular ion of DDANDas more difficult to fragment usefully with low product ion inten-

ities. Noteworthy amongst these from a mechanistic perspective

2.03 97.07 4.12 4.243.48 98.82 0.91 0.92

is m/z of 303 which is generated by loss of the ketone moiety. Themost abundant fragment of CLA was the ion at m/z of 191, corre-sponding to the cyclohexanecarboxylic acid. The highest intensityin the MS/MS spectrum of AODG was the peak at m/z of 269, whichwas generated by loss of the sugar moiety. Low intensity ions werealso found for the product ions of HM5013620, among which themost abundant was m/z of 305 which corresponds to loss of theethylene glycol. For the IS, the highest intensity product ion wasobserved at m/z of 329, which was formed by loss of formaldehyde.Table 1 shows the optimized MS/MS transitions and correspondingMRM parameters of each analyte.

3.2. Chromatography

Different mobile phase compositions (methanol–water acidi-fied with 0.1% acetic acid (v/v), acetonitrile–water acidified with0.1% acetic acid (v/v) and methanol/acetonitrile–water acidifiedwith 0.1% acetic acid (v/v)) with different gradient elution methodswere explored to optimize chromatographic separation of analytes.Compared to methanol or acetonitrile as mobile phase B alone, a 1:1mixture (v/v) of methanol and acetonitrile was found to improvethe chromatographic separation of the analytes. In addition, inorder to minimize the carryover, different wash solutions andwashing times on the syringe and injection valve of autosamplerwere examined and it was found that isopropanol in basic solu-tion (0.1%NH4OH) removed the analytes remaining in the samplingsystem after injection of the highest calibration standard, and thecarryover was less than 20% of the area of LLOQ.

3.3. Sample preparation

A pilot study showed that AND could be stabilized in acidic con-dition by adding a small volume (2% of the total volume) of 10%

formic acid into the biological matrix. Considering the structurediversity of the seven compounds, a simple protein precipita-tion approach was used as the sample preparation procedure andno further concentration procedures were needed to meet the

86 J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88

Table 5Stability data for each analyte (n = 3).

Analyte Nominal conc. (ng/mL) Bench-top stability inplasma(2.5 h on ice-water)

3 cycles freeze/thawstability(−80 ◦C)

Long-term stability(32 days at −80 ◦C)

Autosampler stability(72 h at 8 ◦C)

RE (%) RSD (%) RE (%) RSD (%) RE (%) RSD (%) RE (%) RSD (%)

AND8.00 7.08 3.14 3.71 0.70 0.42 4.06 −5.13 2.31

1600 −1.67 3.50 1.46 4.54 −11.46 0.41 −2.92 2.26

NAND8.00 3.54 2.60 0.83 6.35 3.21 2.52 −5.13 1.27

1600 1.25 2.23 1.88 2.67 −10.42 1.07 −2.50 2.79

DAND8.00 7.46 4.16 0.00 5.20 5.92 4.85 −5.21 4.65

1600 3.54 3.64 8.13 3.61 −10.21 0.80 −1.25 1.90

DDAND20.0 −5.50 2.31 −5.50 5.72 0.33 3.04 −4.00 1.38

1600 9.79 5.53 10.83 1.17 −5.63 2.89 −1.04 4.44

CLA8.00 11.42 2.74 5.12 7.79 −7.50 3.17 −3.29 4.76

1600 3.54 7.30 12.92 2.50 −13.54 2.21 10.00 5.42

AODG8.00 6.29 5.15 −1.63 10.43 −5.46 3.32 −3.54 3.73

1600 2.29 2.54 9.79 2.92 −9.79 3.27 3.96 0.35

HM50136208.00 8.67 3.67 6.00 6.33 −7.96 7.20 −5.42 2.72

1600 3.54 2.98 4.79 3.39 −11.04 1.07 −4.17 1.99

Table 6Hemolyzed plasma effect for each analyte (n = 6).

Analyte Nominal conc. (ng/mL) Measured conc. (ng/mL) RE (%) RSD (%)

AND8 9.18 ± 0.54 14.71 5.93

150 158.17 ± 2.93 5.44 1.851600 1518.33 ± 38.17 −5.10 2.51

NAND8 8.52 ± 0.20 6.48 2.37

150 156.17 ± 2.14 4.11 1.371600 1548.33 ± 44.46 −3.23 2.87

DAND8 8.64 ± 0.45 7.98 5.20

150 153.50 ± 4.23 2.33 2.761600 1691.67 ± 51.15 5.73 3.02

DDAND20 20.82 ± 1.03 4.08 4.97

150 163.17 ± 5.42 8.78 3.321600 1668.33 ± 52.69 4.27 3.16

CLA8 7.50 ± 0.21 −6.21 2.82

150 147.33 ± 2.42 −1.78 1.641600 1563.33 ± 59.22 −2.29 3.79

AODG8 7.81 ± 0.51 −2.38 6.56

150 155.33 ± 2.50 3.56 1.611600 1633.33 ± 57.15 2.08 3.50

HM50136208 8.60 ± 0.28 7.48 3.22

150 159.50 ± 2.81 6.33 1.761600 1603.33 ± 41.79 0.21 2.61

Table 7Pharmacokinetic parameters of all components in SD rats after oral administration of 750 mg/kg HMPL-004 (mean ± SD, n = 6).

Parameters Unit AND NAND DAND DDAND CLA AODG HM5013620

t1/2 h 3.10 ± 1.92 7.99 ± 7.65 3.15 ± 2.20 2.61 ± 2.43 2.88 ± 3.17 5.96 ± 9.69 3.37 ± 1.65MRT h 4.53 ± 1.49 10.62 ± 10.90 7.21 ± 2.54 3.77 ± 3.13 3.39 ± 1.32 11.62 ± 13.06 5.66 ± 2.17AUClast h ng/mL 467.73 ± 102.32 358.48 ± 139.62 232.41 ± 144.07 677.31 ± 261.04 977.88 ± 326.59 613.44 ± 629.80 1378.26 ± 531.08AUCtotal h ng/mL 523.86 ± 134.30 591.79 ± 364.24 291.81 ± 201.35 695.04 ± 187.67 1049.80 ± 283.68 810.21 ± 669.78 1575.12 ± 635.54Cmax ng/mL 180.98 ± 71.16 156.73 ± 95.29 62.10 ± 29.00 386.17 ± 100.64 299.67 ± 100.13 98.87 ± 93.19 366.33 ± 86.63Tmax h 0.29 ± 0.10 0.29 ± 0.10 0.29 ± 0.10 0.29 ± 0.10 1.71 ± 1.35 6.00 ± 2.19 0.67 ± 0.49

J. Wang et al. / J. Chromatogr. B 951–952 (2014) 78–88 87

AND,

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Fig. 4. The average plasma concentration–time curves of AND, NAND, DAND, DD

ensitivity requirements of the method. Cortisone was selected asS because it showed low interference in the biological matrix, hasood stability under the analysis conditions and showed consistentxtraction efficiency.

.4. Validation

.4.1. Specificity and selectivityFig. 3 depicts the typical chromatograms for AND, NAND, DAND,

DAND, CLA, AODG, HM5013620 and IS obtained from the analysisf pre-treated calibration standard samples at the LLOQ and a blanklasma sample. Except for AND and DDAND, no obvious interfer-

nce peaks were observed at the retention times of the analytesn the blank plasma sample. For AND and DDAND, the areas of thenterference peaks in the blank plasma samples were not exceeding% and 16% of the areas at the LLOQ levels, respectively.

CLA, AODG and HM5013620 after a single oral dose of 750 mg/kg of HMPL-004.

3.5. Linearity and LLOQ

Table 2 lists the regression equations of calibration curves andlinearity ranges for all seven analytes. The calibration curves werelinear over the concentration range of 8–2000 ng/mL for DDANDand 4–2000 ng/mL for others. The LLOQ of each analyte was 4 ng/mLwith the exception that the LLOQ of DDAND was 8 ng/mL.

3.6. Accuracy and precision

The intra- and inter-day precision and accuracy values for theQC samples are summarized in Table 3. The intra- and inter-day

precision values of AND, NAND, DAND, DDAND, CLA, AODG andHM5013620 were all less than 12.37%, whilst the accuracy valueswere all within the range of −13.88% to 1.25%. These results indicatethat the method is both accurate and precise.

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8 J. Wang et al. / J. Chroma

.6.1. Extraction recovery and matrix effectThe extraction recoveries and matrix effects of all analytes are

ummarized in Table 4. At three concentration levels, the meanxtraction recoveries were all higher than 90% with RSD less than2% among six replicates, except for AODG and CLA. For AODG, theean extraction recoveries at medium and high concentration lev-

ls (150 and 1600 ng/mL) were around 80% with a RSD less than% and for CLA, the mean extraction recoveries at medium andigh concentration levels (150 and 1600 ng/mL) were around 63%ith a RSD less than 7%. All these data indicate that the extractionethod is efficient and reproducible. The matrix effects of each ana-

yte determined at three concentration levels were all within theange of 85–115%, indicating that no significant influences on theonization of each analyte exist by co-eluted compounds.

.7. Stability

The analytes were stable in plasma stored on ice-water for ateast 2.5 h after acidification with 10% formic acid (For each analyte:E in the range of −5.50% to 11.42% with RSD ≤ 7.30%), in plasmafter three freeze-thaw cycles at −80 ◦C (RE in the range of −5.50%o 12.92% with RSD ≤ 10.43% for each analyte), and in plasma storedt −80 ◦C for 32 days (RE in the range of −13.54% to 5.92% withSD ≤ 7.20% for each analyte). In addition, the analytes were alsoemonstrated to be stable in the pre-treated samples at 8 ◦C for upo 72 h (RE in the range of −5.42% to 10.00% with RSD ≤ 5.42% forach analyte). All these results are summarized in Table 5.

.8. Hemolyzed plasma effect

The effects of hemolysis on AND, NAND, DAND, DDAND, CLA,ODG and HM5013620 are summarized in Table 6. The RSD ofll analytes was no more than 6.56%, whilst RE values were allithin the range of −6.21% to 14.71%, indicating that hemolysisas minimal effect on the quantification of all these seven analytes.

.9. Method application

The validated method was successfully applied to simulta-eously determine the plasma concentrations of AND, NAND,AND, DDAND, CLA, AODG and HM5013620 (the major circulat-

ng metabolite of AND) in SD rats after a single oral administrationf 750 mg/kg of HMPL-004, which is equivalent to 48.75 mg/kg ofND, 23.25 mg/kg of NAND, 3.00 mg/kg of DAND, 25.50 mg/kg ofDAND, 14.25 mg/kg of CLA and 7.50 mg/kg of AODG. The meanlasma concentration-time curve of each analyte is shown inig. 4 and the pharmacokinetic parameters were calculated byon-compartmental analysis (NCA) using Thermo KineticaTM 4.4.1Thermo Electron Corporation) and listed in Table 7.

As the representative chemical components of HMPL-004, allnalytes were detectable until 12 h post dose except for AND, whichas detectable up to 24 h post dose. HM5013620, the hydrolysisetabolite of AND, showed the highest plasma exposure (AUC,

he area under the curve) among all seven analytes. HM5013620as therefore been shown to be one of the major metabolites ofND with about 3-fold higher AUC than its parent compound in

he circulating system. Several studies have reported that phaseI conjugated metabolites were the major metabolites of AND inhe excreta [8–10]. Until now, no reports of circulating metabo-ites of AND have been found. In this study, a hydrolysis metabolitef AND was found in the circulating system and quantitativelynalyzed in plasma samples. Our previous studies have shown

hat few of HM5013620 was generated after incubation with liver

icrosomes or S9s, indicating that it was possibly formed via non-epatic enzymes such as the esterase existing in the blood (Dataot shown).

[

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951–952 (2014) 78–88

In the past decades, a few papers have reported methods toquantitatively determine AND in animals and humans [11–14].We find it surprising that no one has ever realized the instabil-ity of AND in plasma, which led to the possible underestimationof the exposure to AND. In this study, AND was found to be stablein plasma under acidic condition on ice-water for at least about2.5 h, which was long enough for the whole period of samplepre-treatment. Using the current method, the exposure of AND fol-lowing dose normalization was much higher than the previouslyreported data (523.86 h ng/mL at 43.75 mg/kg versus reference dataof 490.83 h ng/mL at 120 mg/kg [14]).

AND, NAND, DAND and DDAND have already been shown to bethe major diterpenoid components in A. paniculata, but until now,we have been unaware of methods to simultaneously determinethese four components.

4. Conclusion

A simple LC–MS/MS method for the simultaneous quantifi-cation of six chemical components of HMPL-004 (AND, NAND,DAND, DDAND, CLA and AODG) and one major metabolite of AND(HM5013620) in rat plasma was developed and validated in thecurrent study and proven to be highly robust, accurate, sensitiveand specific. In this study, AND was found to be unstable in plasma,but could be stabilized via acidification. In addition, the hydrolysismetabolite of AND, named HM5013620, was found to be the majorcirculating metabolite with 3-fold higher exposure than AND inrat plasma. The developed method has been applied successfullyin the rat pharmacokinetic study and will be favorable for the fur-ther studies of HMPL-004 and other pharmacokinetic studies of A.paniculata related TCMs.

Acknowledgements

The author would like to thank Dr. Weihan Zhang, the seniordirector of the Medicinal Chemistry group of HMP, and Dr. ZhenpingWu, the senior vice president and group leader of the Pharmaceu-tical Science group of HMP, for providing the standard materials ofall analytes and also thank all other colleagues in HMPL-004 projectgroups of HMP for the support on this study.

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