MS and its application

Journal of Chromatography B, 1007 (2015) 81–92

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Highly sensitive method for simultaneous determination of nine alkaloids of Shuanghua Baihe tablets in human plasma by LC–MS/MS and its application Yao Wu a,b,1 , Ruijuan Liu a,b,1 , Pan Gu a,b , Minlu Cheng a,b , Lu Zheng c , Yujie Liu c , Pengcheng Ma d , Li Ding a,b,∗ a

Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China Nanjing Clinical Tech Laboratories Inc., 18 Zhilan Road, Jiangning District, Nanjing 211000, PR China c Yangtze River Pharmaceutical Group, Taizhou 225321, PR China d Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, 12 Jiangwangmiao Street, Nanjing 210042, PR China b

a r t i c l e

i n f o

Article history: Received 9 May 2015 Received in revised form 10 October 2015 Accepted 14 October 2015 Available online 7 November 2015 Keywords: Shuanghua Baihe tablets LC–MS/MS Berberine Palmatine Coptisine Corynoline

a b s t r a c t Shuanghua Baihe tablets (SBT) is a traditional Chinese medicinal formula which has been used to treat recurrent aphthous stomatitis for many years. To study the pharmacokinetic profiles of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline in human after administration of SBT, a sensitive liquid chromatography–tandem mass spectrometry method was developed and fully validated for the simultaneous quantification of these nine alkaloids in human plasma. After protein precipitation, the nine alkaloids in human plasma sample was separated on a Hanbon C18 (150 mm × 2.1 mm, 5 ␮m) column with gradient elution using methanol and 0.5% formic acid water solution, and detected by a triple quadrupole mass spectrometer with an electrospray ionization source. It is a challenge to design different calibration ranges for different analytes in a bioanalytical method for simultaneous determination of multi-analytes in bio-samples. To ensure that each alkaloid in the plasma was determined accurately by the simultaneous quantitation method, the upper limits of quantification for the nine alkaloids were designed at 100, 300, 800, 1800 and 5000 pg/mL, respectively, according to the maximum plasma concentration value of each alkaloid obtained from the pilot pharmacokinetic study. The lower limit of quantification was 15 pg/mL for berberine, epiberberine, coptisine, magnoflorine, berberrubine, corynoline and acetylcorynoline, while for palmatine and jatrorrhizine it was 1.5 pg/mL. This method was successfully applied to investigate the pharmacokinetic profiles of the nine alkaloids in healthy Chinese volunteers after a single oral administration of SBT. © 2015 Published by Elsevier B.V.

1. Introduction Shuanghua Baihe tablets (SBT), a herbal formula composed of Coptidis Rhizoma, Corydalis Bungeanae, Isatids Radix and Arnebiae Radix, et al., has been selected to treat recurrent aphthous stomatitis (RAS) for years in China. RAS, which is classified as aphtha

Abbreviations: SBT, Shuanghua Baihe tablets; RAS, recurrent aphthous stomatitis; TCM, traditional Chinese medicine; TCMs, traditional Chinese medicines; LLOQ, lower limit of quantification; IS, internal standard; MRM, multiple reaction monitoring; PPT, protein precipitation; LLE, liquid–liquid extraction. ∗ Corresponding author at: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. Fax: +86 25 8327 1485. E-mail address: [email protected] (L. Ding). 1 Yao Wu and Ruijuan Liu are first co-authors. http://dx.doi.org/10.1016/j.jchromb.2015.10.015 1570-0232/© 2015 Published by Elsevier B.V.

by traditional Chinese medicine (TCM), is characterized by inflammation with circumscribed recurrent round or oval ulcers covered with a white or yellow pseudomembrane [1]. RAS is very common, affecting all ages and geographical areas, and the morbidity among the general population reaches up to 25% [2]. The oral ulcers may be painful when patients are eating or speaking, which could decrease the quality of life. SBT is effective in treating RAS and the major ingredients in rat plasma and urine after oral administration of SBT have been identified [3], but the pharmacokinetic studies of SBT in human remain scanty. Heat accumulation in heart and spleen has been regarded as the leading etiology of RAS in the perspective of TCM. The investigations of modern medicine illustrate that microorganisms, such as Helicobacter pylori, may associate with RAS [4–6]. The development of SBT is based on the TCM theory of treating heat

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Fig. 1. Chemical structures and positive product ion mass spectra of berberine (a), epiberberine (b), coptisine (c), palmatine (d), jatrorrhizine (e), magnoflorine (f), berberrubine (g), corynoline (h), acetylcorynoline (i) and donepezil (internal standard, j).

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Fig. 2. Representative multiple reaction monitoring chromatograms of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline, acetylcorynoline and donepezil (internal standard). (a) blank plasma; (b) blank plasma spiked with the analytes (at medium concentration level); (c) 2.0 h sample plasma after a single oral administration of Shuanghua Baihe tablets.

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Fig. 3. Mean plasma concentration-time profiles of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline in human plasma after a single oral administration of Shuanghua Baihe tablet (n = 2).

Table 1 Multiple reaction monitoring transitions and correlative optimized parameters used for the detection. Analytes

Berberine Epiberberine Coptisine Palmatine Jatrorrhizine Magnoflorine Berberrubine Corynoline Acetylcorynoline Donepezil (IS)

Transition Precursor ion

Product ion

336.1 336.1 320.1 352.2 338.1 342.2 322.0 368.1 410.2 380.2

320.1 320.1 292.2 336.2 322.2 297.2 307.2 289.1 289.2 243.2

Dwell (ms)

DP (V)

EP (V)

CE (V)

CXP (V)

500 500 500 500 500 500 500 500 500 500

45 45 19 25 60 37 30 15 34 68

13.0 13.0 3.0 8.5 11.5 9.0 5.0 4.5 13.0 7.4

42.0 42.0 46.5 38.0 38.0 35.0 37.5 34.0 35.0 43.5

27 27 40 23 7 36 28 23 25 20

DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: collision cell exit potential.

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Table 2 Calibration curves and linear range of the nine alkaloids. Analytes

Calibration curve

r

Linear range (pg/mL)

Berberine Epiberberine Coptisine Palmatine Jatrorrhizine Magnoflorine Berberrubine Corynoline Acetylcorynoline

f = 3.29 × C + 28.5 f = 7.65 × C + 24.3 f = 2.93 × C + 2.14 f = 19.3 × C + 1.72 f = 23.9 × C + 21.6 f = 1.94 × C + 1.67 f = 20.6 × C − 249 f = 6.42 × C + 61.4 f = 1.42 × C + 4.94

0.9986 0.9960 0.9953 0.9966 0.9992 0.9979 0.9987 0.9974 0.9979

15.61–832.7 15.93–318.7 16.52–881.2 1.451–96.77 1.594–106.3 14.75–4916 15.18–5060 11.90–1785 14.73–785.7

Table 3 The precision and accuracy values for quantification of the nine alkaloids in human plasma (n = 5). Analytes

Spiked concentration (pg/mL)

Measured concentration (mean ± SD, pg/mL)

Berberine

15.61 (LLOQ) 46.84 208.2 624.6 15.93 (LLOQ) 47.80 106.2 265.6 16.52 (LLOQ) 49.57 220.3 660.9 1.451 (LLOQ) 3.871 19.35 77.41 1.594 (LLOQ) 4.250 21.25 85.00 14.75 (LLOQ) 44.25 393.3 3933 15.18 (LLOQ) 45.54 404.8 4048 11.90 (LLOQ) 53.55 238.0 1428 14.73 (LLOQ) 44.20 196.4 589.3

14.98 ± 1.91 45.68 ± 4.75 220.0 ± 16.9 647.6 ± 43.5 14.74 ± 1.40 44.86 ± 5.38 106.5 ± 7.2 275.9 ± 23.5 15.44 ± 1.53 51.48 ± 5.59 219.0 ± 22.8 674.9 ± 47.1 1.429 ± 0.186 3.952 ± 0.343 20.18 ± 1.69 77.56 ± 5.03 1.682 ± 0.200 4.296 ± 0.343 22.27 ± 1.52 91.00 ± 5.38 14.25 ± 1.52 45.97 ± 4.27 366.6 ± 33.0 4187 ± 296 17.31 ± 0.66 46.41 ± 3.28 366.4 ± 22.1 4070 ± 232 13.60 ± 0.98 53.05 ± 3.61 249.8 ± 10.4 1534 ± 56 16.21 ± 1.78 43.17 ± 5.44 183.9 ± 13.6 564.4 ± 46.3

Epiberberine

Coptisine

Palmatine

Jatrorrhizine

Magnoflorine

Berberrubine

Corynoline

Acetylcorynoline

syndrome with cold-natured drugs and treating excess syndrome with purgative method. The formula of SBT mainly consists of Coptidis Rhizoma, as the emperor herb, and Corydalis Bungeanae that are used to clear damp-heat and cool blood. Recently, it has been proved that berberine, the major component of Coptidis Rhizoma, plays a significant role in the anti-inflammatory effects [7] and bacteriostasis [8,9]. As for other ingredients of Coptidis Rhizoma, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine and berberrubine, it has been reported that they have anti-microbial or anti-inflammatory properties [10–14]. Corydalis Bungeanae, which shows various pharmacological effects such as anti-inflammation and bacteriostasis [15], contains corynoline and acetylcorynoline as the main active ingredients. Studies on the pharmacokinetics of the key components are important for the proper application of SBT in clinic. To investigate the fate of the active components contained in traditional Chinese medicines (TCMs) in vivo, the methods

RSD (%)

RE (%)

Intra-day

Inter-day

11.1 11.1 5.8 4.4 7.1 12.3 4.5 8.5 7.1 10.5 9.5 2.5 12.8 8.1 6.7 5.3 12.8 7.0 4.9 4.5 11.0 9.8 8.4 7.1 2.4 4.7 4.1 4.2 7.4 4.4 4.4 3.8 9.6 12.6 7.5 8.0

18.9 4.5 14.4 14.1 18.2 9.8 14.2 8.7 18.7 12.9 13.6 13.5 14.1 11.6 13.9 11.4 2.9 12.5 13.4 11.1 8.0 5.1 12.2 6.9 8.2 13.8 12.4 11.0 6.3 14.4 2.4 1.7 17.1 12.4 6.6 9.1

−4.0 −2.5 5.7 3.7 −7.5 −6.2 0.3 3.9 −6.5 3.9 −0.6 2.1 −1.5 2.1 4.3 0.2 5.5 1.1 4.8 7.1 −3.4 3.9 −6.8 6.5 14.0 1.9 −9.5 0.5 14.3 −0.9 5.0 7.4 10.0 −2.3 −6.4 −4.2

development for biopharmaceutical analysis focused on these components is becoming more and more popular recently [16–20]. TCMs have been widely used in China for thousands of years, but the pharmacokinetic profiles of their active components in human are rare, including SBT. In recent years, the TCM scientists are more keenly aware that the pharmacokinetic characteristics of the active components contained in TCMs is very important for the reasonable use of TCMs in clinic and explanation of their pharmacological effects. The main active components in SBT are isoquinoline alkaloids called berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline. Sponsored by Yangtze River Pharmaceutical Group, the manufacturer of SBT, a pharmacokinetic study of these nine alkaloids in human after oral administration of SBT was carried on in our laboratories. To evaluate the pharmacokinetics of these nine main alkaloids in SBT in human, a sensitive method for the simultaneous determination of these alkaloids in

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Table 4 The test results of the recovery and normalized matrix effect in plasma. Analytes

Added (pg/mL)

Recovery (%, n = 5)

Berberine

46.84 208.2 624.6 47.80 106.2 265.6 49.57 220.3 660.9 3.871 19.35 77.41 4.250 21.25 85.00 44.25 393.3 3933 45.54 404.8 4048 53.55 238.0 1428 44.20 196.4 589.3 668.0

91.2 85.7 86.2 96.6 95.2 95.7 87.0 83.9 78.8 87.3 78.4 79.4 87.6 87.3 92.5 83.3 84.6 82.0 77.4 81.4 86.6 84.0 85.6 86.8 77.1 83.9 86.9 88.1

Epiberberine

Coptisine

Palmatine

Jatrorrhizine

Magnoflorine

Berberrubine

Corynoline

Acetylcorynoline

Donepezil (IS)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.1 2.9 4.2 6.0 4.6 8.6 8.4 4.8 4.7 3.3 4.7 3.7 4.7 3.1 4.4 3.3 3.1 3.9 6.6 1.8 8.5 5.9 0.7 1.1 10.4 7.6 5.8 5.3

human plasma is critical. To date, several reliable methods have been reported for the determination of some isoquinoline alkaloids like berberine, epiberberine, coptisine, palmatine, jatrorrhizine and magnoflorine in animal plasma [21–24], and applied for the animal studies only. Among these methods, the most sensitive one has the lower limit of quantification (LLOQ) of 0.1 ng/mL, which is not sensitive for the pharmacokinetic study of these alkaloids in human. The LLOQ at pg/mL level of the developed method should be reached for the pharmacokinetic study of the nine alkaloids of SBT in human plasma. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline in human plasma with the LLOQs of 1.5 or 15 pg/mL for the nine alkaloids, and successfully applied in the pharmacokinetic study of SBT in health volunteers. 2. Experimental 2.1. Chemicals and reagents The reference standards of berberine (purity 86.7%), palmatine (purity 86.6%), jatrorrhizine (purity 90.3%), corynoline (purity 100%) and donepezil (internal standard, IS, purity 100%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Epiberberine (purity 98%), coptisine (purity 98%), magnoflorine (purity 98%) and berberrubine (purity 98%) were obtained from Shanghai Youxuan Biotechnology Co., Ltd. (Shanghai, China). Acetylcorynoline (purity 98%) were purchased from Chengdu Ruifensi Biotechnology Co., Ltd. (Chengdu, China). SBT (Batch No. 14032311) was supplied by Yangtze River Pharmaceutical Group Co., Ltd. (Taizhou, China), in which the contents of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline were 18.01, 5.38, 5.11, 4.92, 2.42, 1.90, 1.04, 0.47 and 0.06 mg per tablet respectively. HPLC-grade acetonitrile and methanol were purchased from Merck KGaA (Darmstadt, German). Formic acid (Nanjing Chemical Reagent Co., Ltd., Nanjing, China) was of AR

RSD (%)

Normalized matrix effect (%, n = 6)

RSD (%)

6.7 3.4 4.9 6.2 4.8 9.0 9.7 5.7 6.0 3.8 6.0 4.7 5.4 3.6 4.8 4.0 3.7 4.8 8.5 2.2 9.8 7.0 0.8 1.3 13.4 9.0 6.7 6.0

111.2 ± 3.8

3.4

106.4 ± 7.2 94.5 ± 9.0

6.7 9.5

108.0 ± 5.2 104.8 ± 12.2

4.8 11.6

94.8 ± 9.3 98.5 ± 9.7

9.8 9.8

90.3 ± 3.7 108.3 ± 10.0

4.1 9.2

93.8 ± 2.2 106.9 ± 10.1

2.3 9.5

95.7 ± 3.7 107.3 ± 8.5

3.9 7.9

98.6 ± 3.3 103.6 ± 10.1

3.3 9.7

103.3 ± 8.7 100.0 ± 5.8

8.4 5.8

110.8 ± 4.7 –

4.2 –

grade. Deionized water was produced by a Milli-Q Reagent Water System (Millipore, Bedford, MA, USA). 2.2. Instrumentations and conditions The analytical system included an Agilent 1260 series high performance liquid chromatography (Agilent Technologies, USA) and an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, USA) which were connected by an electrospray ionization (ESI) source. A Hanbon C18 column (150 mm × 2.1 mm, 5 ␮m; Hanbon Science and Technology, China) was used to achieve the separation of various alkaloids at the temperature of 30 ◦ C. The mobile phase, with a flow rate of 0.4 mL/min, consisted of methanol (A) and 0.5% (v/v) formic acid aqueous solution (B). The gradient elution was adopted as follows: 0–1.5 min, 68% B; 1.5–1.8 min, 68–60% B; 1.8–8.5 min, 60% B; 8.5–9.0 min, 60–0% B; 9.0–11.0 min, 0% B; 11.0–11.5 min, 0–68% B; 11.5–20.0 min, 68% B. Methanol/water (1:1, v/v) was selected as the needle rinse solution. The volume of injection was 15 ␮L and the autosampler temperature maintained at 4 ◦ C. Mass spectrometer detector was set as multiple reaction monitoring (MRM) in the positive ion mode. For each analyte, the precursor–product ion pair and corresponding parameters are listed in Table 1. Mass spectrometer conditions were optimized as follows: ion spray temperature was 450 ◦ C; ion spray voltage was 5500 V; nebulizer gas and heater gas were kept at 40 and 50 psi, respectively; curtain gas was 30 psi and collision gas was 12 psi. All the parameter control and data analysis were conducted by AB SCIEX Analyst software (version 1.5.2). 2.3. Stock and standard solutions The stock solutions of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline were prepared in methanol. Working solutions were obtained by serially diluting the stock solutions with methanol/water (1:1, v/v) separately. Then, the corresponding working solutions were mixed to prepare the mixed working solutions over the concentration ranges of

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0.4683–24.98 ng/mL for berberine, 0.4779–9.561 ng/mL for epiberberine, 0.4956–26.44 ng/mL for coptisine, 0.04354–2.903 ng/mL for palmatine, 0.04782–3.189 ng/mL for jatrorrhizine, 0.4425–147.5 ng/mL for magnoflorine, 0.4554–151.8 ng/mL for berberrubine, 0.3570–53.55 ng/mL for corynoline, and 0.4420–23.57 ng/mL for acetylcorynoline, respectively. A 20.04 ng/mL IS working solution was prepared in methanol. All the solutions were stored at −20 ◦ C until use. 2.4. Calibration and quality control standards A series of calibration standards were made by spiking the mixed working solutions (20 ␮L) and the IS working solution (20 ␮L) in blank human plasma (600 ␮L) to yield concentrations of 15.61, 31.23, 62.46, 156.1, 312.3, 520.5 and 832.7 pg/mL for berberine, 15.93, 31.87, 53.12, 106.2, 159.3, 212.5 and 318.7 pg/mL for epiberberine, 16.52, 33.05, 66.09, 165.2, 330.5, 550.8 and 881.2 pg/mL for coptisine, 1.451, 2.903, 5.806, 14.51, 29.03, 58.06 and 96.77 pg/mL for palmatine, 1.594, 3.188, 6.375, 15.94, 31.88, 63.75 and 106.3 pg/mL for jatrorrhizine, 14.75, 29.50, 59.00, 196.7, 491.6, 1475, 2950 and 4916 pg/mL for magnoflorine, 15.18, 30.36, 60.72, 202.4, 506.0, 1518, 3036 and 5060 pg/mL for berberrubine, 11.90, 35.70, 119.0, 297.5, 595.0, 1190 and 1785 pg/mL for corynoline, and 14.73, 29.47, 58.93, 147.3, 294.7, 491.1 and 785.7 pg/mL for acetylcorynoline. The quality control (QC) samples at three concentrations containing berberine (46.84, 208.2, and 624.6 pg/mL), epiberberine (47.80, 106.2, and 265.6 pg/mL), coptisine (49.57, 220.3 and 660.9 pg/mL), palmatine (3.871, 19.35, and 77.41 pg/mL), jatrorrhizine (4.250, 21.25, and 85.00 pg/mL), magnoflorine (44.25, 393.3, and 3933 pg/mL), berberrubine (45.54, 404.8, and 4048 pg/mL), corynoline (53.55, 238.0, and 1428 pg/mL), acetylcorynoline (44.20, 196.4, and 589.3 pg/mL) and the IS (668.0 pg/mL) were prepared in the same way as the calibration standards. 2.5. Sample preparation Plasma samples were thawed at room temperature and thoroughly vortexed prior to analysis. To aliquot of 600 ␮L of plasma samples, 20 ␮L of the IS working solution were added and vortexmixed for 10 s. Then the mixture was precipitated with 800 ␮L acetonitrile and 400 ␮L methanol, vortex-mixed for 3 min, followed by centrifuging at 15600 rpm for 10 min. The supernatant was separated and evaporated to dryness with nitrogen at 40 ◦ C. The residue was reconstituted in 150 ␮L initial mobile phase, vortexed for 3 min, and then centrifuged at 15600 rpm for 10 min. The supernatant was transferred to the autosampler vial and an aliquot of 15 ␮L was injected into the LC–MS/MS system for analysis. 2.6. Method validation The method validation was performed based on the recently published guidelines of the United States Food and Drug Administration (FDA) for bioanalytical method validation [25]. 2.6.1. Selectivity The selectivity of this method was assessed by comparing chromatograms from six different batches of drug-free human plasma sample, blank plasma sample spiked with all the analytes, and a human plasma sample obtained from a subject after oral administration of SBT. 2.6.2. Linearity and sensitivity There were seven concentration levels on the calibration curves of berberine, epiberberine, coptisine, palmatine, jatrorrhizine,

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corynoline and acetylcorynoline, and eight levels on that of magnoflorine and berberrubine. Each sample for calibration curves was freshly prepared and assayed on three independent days. Blank plasma samples and drug-free plasma spiked with the IS were also analyzed to confirm the absence of interferences. Linearity was investigated by the plot of the response ratios of analytes versus the IS against the concentrations of the calibration standards, and the calibration curves were fitted applying a weighted (1/x2 ) leastsquares linear regression method. The LLOQs, which are defined as the lowest concentration on the standard curves, represent the sensitivity of the method. The analytes response at the LLOQ samples should be detected with acceptable accuracy and precision, and be detected as signal-to-noise ratio ≥ 5.

2.6.3. Accuracy and precision The accuracy and precision were evaluated over two days by the quantification of three validation batches, each consisting of five replicated QC samples at three concentration levels (low, medium and high). The precision of the method including intra- and interday precision was calculated as the relative standard deviation (RSD) and required to be less than 15%. Accuracy was expressed as the relative error (RE) which was required to be within ±15%.

2.6.4. Extraction recovery and matrix effect Five replicates of QC samples at each of the concentration levels (low, medium and high) were prepared to assess recovery efficiency of the analytes. Recovery was expressed as the ratio of peak area obtained from the extracted spiked matrix to peak area of the equivalent blank plasma samples spiked after the extraction. The matrix effect was evaluated by assaying six individual QC samples at low and high concentration levels and was calculated as the ratio of peak areas of the post-extraction spiked samples to that of the analytes resolved in mobile phase. Considering the matrix effect of the IS, normalized matrix factors determined by comparing the matrix factor of analytes with the matrix factor of the IS were used to evaluate the effects of matrix on ionization.

2.6.5. Carryover effect Carryover effect was assessed by injecting the processed blank samples after injecting upper limit of the quantification (ULOQ) samples in five repeat runs. It is required that the response in the blank matrix at the retention times of the analytes should be less than 10% of the response of LLOQ samples.

2.6.6. Stability The stability tests were carried out on the low and high QC concentration levels for all the analytes in plasma after storage at room temperature for 8 h, −20 ◦ C for 65 days, and three freeze (−20 ◦ C)–thaw (room temperature) cycles. The post-preparative stability of QC samples was tested by reanalyzing the prepared samples kept in autosampler at 4 ◦ C for 29 h. And the extracted residues of QC samples kept under room temperature for 24 h, and −20 ◦ C for 10 days were used to evaluate the stability of the extracted residues.

2.6.7. Dilution integrity It is necessary to assess the dilution integrity for these alkaloids whose plasma concentration was above the upper limits of their standard curves. The highly concentrated samples were 5-fold diluted with blank plasma prior to extraction and assayed along with corresponding QC samples at low and high concentration levels in a validation run.

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Table 5 Stability test results of the nine alkaloids under various storage conditions (n = 3). Analyte

Added (pg/mL)

Epiberberine Coptisine Palmatine Jatrorrhizine Magnoflorine Berberrubine Corynoline Acetylcorynoline

46.84 624.6 47.80 265.6 49.57 660.9 3.871 77.41 4.250 85.00 44.25 3933 45.54 4048 53.55 1428 44.20 589.3

43.55 605.1 42.60 263.6 47.15 644.0 3.392 73.13 4.711 89.05 45.62 4371 46.27 4240 60.20 1558 39.38 508.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.74 44.1 4.12 26.4 4.04 93.0 0.378 2.43 0.211 8.74 1.59 32 4.55 83 1.10 38 5.62 28.0

RE (%)

Measured (pg/mL)

−7.0 −3.1 −10.9 −0.8 −4.9 −2.6 −12.4 −5.5 10.9 4.8 3.1 11.1 1.6 4.8 12.4 9.1 −10.9 −13.7

49.15 627.6 47.59 238.7 47.54 626.5 3.881 80.76 4.357 74.69 44.71 3819 41.46 4559 49.70 1279 47.14 554.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.03 10.3 4.99 7.4 2.74 24.3 0.397 1.99 0.482 1.53 2.98 130 0.88 27 3.18 5 2.19 34.7

After three freeze–thaw cycles RE (%)

Measured (pg/mL)

4.9 0.5 −0.4 −10.1 −4.1 −5.2 0.3 4.3 2.5 −12.1 1.0 −2.9 −9.0 12.6 −7.2 −10.4 6.6 −6.0

51.20 667.1 50.26 278.0 54.54 678.9 3.339 76.76 4.642 96.76 50.41 3670 45.00 3712 50.36 1596 40.89 527.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.49 20.6 0.06 8.6 2.05 58.7 0.343 10.84 0.657 0.43 2.39 154 0.23 31 2.11 8 2.51 22.0

Residue stability (room temperature, 24 h)

Residue stability (−20 ◦ C, 10 days)

RE (%)

Measured (pg/mL)

RE (%)

Measured (pg/mL)

10.0 −5.1 11.8 −7.9 4.0 −12.7 2.1 8.9 2.4 −5.4 9.5 0.3 9.8 2.8 −4.5 0.4 −10.6 −13.4

46.05 613.1 46.33 269.7 45.29 658.6 3.741 76.96 4.520 88.39 46.49 4314 49.80 4098 60.48 1563 39.21 510.4

−1.7 −1.8 −3.1 1.5 −8.6 −0.4 −3.3 −0.6 6.4 4.0 5.1 9.7 9.4 1.2 12.9 9.4 −11.3 −13.4

43.54 668.1 42.98 287.0 53.83 732.8 4.296 87.74 4.337 96.05 44.98 3792 42.50 3609 46.23 1543 46.86 632.7

Autosampler stability (4 ◦ C, 29 h) RE (%)

Measured (pg/mL)

9.3 6.8 5.2 4.7 10.0 2.7 −13.7 −0.8 9.2 13.8 13.9 −6.7 −1.2 −8.3 −6.0 11.8 −7.5 −10.6

51.50 592.8 53.44 244.6 51.55 577.2 3.952 84.30 4.351 80.39 48.46 3944 49.99 4161 51.14 1433 39.52 510.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.13 61.2 1.18 20.0 0.79 63.5 0.329 1.50 0.153 2.66 2.99 106 3.82 118 1.66 28 1.58 6.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.18 41.2 6.36 3.2 2.18 48.5 0.181 1.76 0.561 3.90 4.23 20 1.87 53 0.45 61 1.41 25.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.44 23.0 2.27 13.0 1.02 34.2 0.103 0.60 0.168 1.97 3.18 60 1.96 111 4.00 14 5.24 15.8

RE (%) −7.0 7.0 −10.1 8.0 8.6 10.9 11.0 13.3 2.0 13.0 1.6 −3.6 −6.7 −10.9 −13.5 8.1 −13.5 7.4

Y. Wu et al. / J. Chromatogr. B 1007 (2015) 81–92

Measured (pg/mL) Berberine

At −20 ◦ C for 65 days

At room temperature for 8 h

Y. Wu et al. / J. Chromatogr. B 1007 (2015) 81–92

89

Table 6 Precision and accuracy of the dilution quality control samples (n = 5). Analytes

Added (pg/mL)

Dilution factor

Caculated (pg/mL)

Measured (pg/mL)

Epiberberine

239.0 1063 551.0 2754 48.39 483.9 2024 10120 1190 7140 491.1 294.7

5

47.80 212.5 110.2 550.8 9.677 96.77 404.8 2024 238.0 1428 98.22 589.3

44.63 201.4 107.1 478.5 8.976 97.21 355.3 2021 213.3 1305 92.99 548.4

Coptisine Palmatine Berberrubine Corynoline Acetylcorynoline

5 5 5 5 5

± ± ± ± ± ± ± ± ± ± ± ±

6.45 24.3 11.3 40.8 0.505 11.91 42.2 98 7.0 154 10.45 44.4

RSD (%)

RE (%)

14.5 12.0 10.5 8.5 5.6 12.3 11.9 4.9 3.3 11.8 11.2 8.1

−6.6 −5.2 −2.8 −13.1 −7.2 0.5 −12.2 −0.2 −10.4 −8.6 −5.3 −6.9

Table 7 Mean pharmacokinetic parameters of the nine alkaloids in human plasma. Analytes

Cmax (pg/mL)

Tmax (h)

t1/2 (h)

AUC0−96 (pg h/mL)

Berberine Epiberberine Coptisine Palmatine Jatrorrhizine Magnoflorine Berberrubine Corynoline Acetylcorynoline

281.0 183.3 529.7 45.41 18.25 2584 3522 600.8 386.7

1.34 1.67 1.92 1.50 2.67 3.00 1.09 1.34 1.75

31.52 45.04 45.60 22.36 22.22 19.26 19.23 2.02 4.13

2994 931.3 6479 504.0 269.5 27018 15026 1666 971.3

2.7. Clinical application The pharmacokinetic study was submitted to the Medical Ethic Committee of the Institute of Dermatology affiliated to Chinese Academy of Medical Science (Nanjing, China). Two healthy Chinese volunteers, one male (weight of 86 kg, height of 186 cm, age of 20) and one female (weight of 55 kg, height of 163 cm, age of 21), were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. They were examined to be healthy on the basis of medical history, physical examination, laboratory examination and ECG. They administered a single dose of 4 tablets of SBT with 250 mL water after an overnight fast at least of 10 h. Water intake was allowed 2 h after administration, and standard meals were provided 6 h after administration. A 5 mL blood sample was drawn into a heparinized and labeled tube before drug administration (0 h) as well as 0.25, 0.5, 0.75, 1, 1.33, 1.67, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 48, 72 and 96 h post-dosing. Then the blood samples were immediately centrifuged for 5 min, and the plasma fractions were separated and stored at −20 ◦ C until analysis. The concentrations of the nine alkaloids in plasma were determined by this validated liquid chromatography–mass spectrometry method. The pharmacokinetic parameters were calculated by DAS 3.2.7 (DAS® ; professional edition version 3.2.7, Drug and Statistics, Shanghai, China) using non-compartmental methods. The pharmacokinetic parameters studied in this study were maximum plasma concentration (Cmax ), the time to Cmax (Tmax ), elimination half-life (t1/2 ), the area under the plasma concentration-time curve from 0 to t (AUC0−t ). 3. Results and discussion 3.1. Design for calibration range It is a challenge to design different calibration ranges for different analytes in a bioanalytical method for simultaneous determination of multi-analytes in bio-samples. The design of ULOQs possesses the same importance as that of LLOQs. If the ULOQs are set too high, the precision and accuracy of the samples at low con-

centration levels would be badly affected. According to the results of the pilot pharmacokinetic study, the maximum concentration values of the nine alkaloids in human plasma after the administration of SBT varied considerably. For example, the maximum plasma concentration value was 31.90 pg/mL for jatrorrhizine, while for berberrubine it was 3.300 ng/mL which was about 103 times higher than that of jatrorrhizine. To ensure that the concentration values of all the analytes in the plasma samples were determined accurately by the simultaneous quantification method, the ULOQs were designed at 100 pg/mL for palmatine and jatrorrhizine, 300 pg/mL for epiberberine, 800 pg/mL for berberine, coptisine and acetylcorynoline, 1800 pg/mL for corynoline, 5000 pg/mL for magnoflorine and berberrubine, respectively. The data of the pilot pharmacokinetic study show that the minimum concentration values at the terminal phase of the plasma concentration-time profiles were about 20 pg/mL for berberine, epiberberine, coptisine, magnoflorine, berberrubine, corynoline and acetylcorynoline and were about 2 pg/mL for palmatine and jatrorrhizine. To satisfy the requirements of pharmacokinetic study, the LLOQs for the nine alkaloids were set at 1.5 and 15 pg/mL, respectively, according to the minimum plasma concentration value for each alkaloid obtained from the pilot pharmacokinetic study.

3.2. Development of extraction condition In the process of sample preparation, protein precipitation (PPT) and ethyl acetate liquid–liquid extraction (LLE) were compared. The results indicated that the recovery of PPT for most of these alkaloids including berberine, epiberberine, coptisine, palmatine, jatrorrhizine and magnoflorine was higher than those of LLE, because of the existence of the quaternary nitrogen in their chemical structures. Therefore, PPT was selected for the extraction of the samples. To avoid the matrix effect and obtain the higher extraction efficiency, different kinds of precipitants including acetonitrile, methanol, methanol/acetonitrile (2:1, v/v) and methanol/acetonitrile (1:2, v/v) were tested. Finally, the methanol/acetonitrile (1:2, v/v) was selected because it yielded rel-

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atively high recovery efficiency and got rid of the interference from the endogenous substances for each analyte. 3.3. LC–MS/MS optimization 3.3.1. Mass spectrometry A positive ion-monitoring mode with ESI was adopted to determine the concentration values of the target alkaloids. The standard solutions were respectively infused into the mass spectrometer to ascertain their precursor ions and to select product ions for the application in MRM mode. The predominant ions in the full scan mass spectra were selected as the parent ions for the analytes. For the six quaternary ammonium alkaloids, the parent ions were quaternary ammonium ions [M]+ at m/z 336.1, 336.1, 320.1, 352.2, 338.1 and 342.2 for berberine, epiberberine, coptisine, palmatine, jatrorrhizine and magnoflorine, respectively. The protonated molecular ions [M + H]+ at m/z 322.0, 368.1, 410.2 and 380.2 were chosen for berberrubine, corynoline, acetylcorynoline and the IS, respectively. The predominant ions in the product ion scan mass spectra were also selected as the product ions for the analytes in MRM analysis. They were the fragment ions at m/z 320.1, 320.1, 292.2, 336.2, 322.2, 297.2, 307.2, 289.1, 289.2 and 243.2 for berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline, acetylcorynoline and the IS, respectively. Fig. 1 shows the MS/MS product ion spectra of the analytes. Meanwhile, the ion spray temperature, ion spray voltage, nebulizer gas, heater gas, curtain gas and collision gas were optimized to obtain the higher sensitivity. 3.3.2. Liquid chromatographic conditions The ratio of the organic portion in the initial mobile phase should be low to separate magnoflorine, which was weakly retained on the C18 column, from early-eluted endogenous compounds and overcome matrix effect on it. The results showed that methanol/water (32:68, v/v) was a proper initial mobile phase ratio for the separation of magnoflorine. However, the retention of the other eight alkaloids on the C18 column was much higher than that of magnoflorine. To elute these alkaloids at proper time, the ratio of organic portion of the mobile phase should be increased, so a gradient elution system was investigated for the sample separation. The initial mobile phase ratio was kept for 1.5 min for the separation of magnoflorine, and the water portion ratio of the mobile phase begin to decrease slowly for the elution of the other eight alkaloids. Considering that berberine and epiberberine were isomers and were detected using the same precursor-product ion transition, the absolutely separation for these two analytes from each other is needed. Therefore, while the ratio of the water portion of the mobile phase was decreased to 60% at 1.8 min post sample injection, the decrease of the water portion ratio was stopped and the ratio of 60% was maintained to 8.5 min post sample injection to let berberine and epiberberine be separated from each other. Fortunately, the other six alkaloids were also properly eluted during the time period of 1.8–8.5 min post sample injection. After all of these alkaloids were eluted from the column, the water portion of the mobile phase was decreased to 0%, and the column was washed with the pure methanol for 2 min. In the selection of the gradient elution program, we found that the acid condition could reduce the matrix effect, and the different addition amounts of formic acid were tested for the matrix effect. The results showed that the addition of 0.5% formic acid to the aqueous portion of the mobile phase could overcome the matrix effect on the analytes significantly, so 0.5% formic acid aqueous solution was finally used as the water potion of the mobile phase. In order to avoid the late-eluted matrix components to interfere the nine analytes of the next injections [26], the ratio of methanol in the mobile phase was finally increased to 100% after all

the nine alkaloids were eluted, and the column was washed with the pure methanol for 2 min. 3.4. Method validation 3.4.1. Selectivity The typical chromatograms of the blank plasma sample, blank plasma sample spiked with all the analytes, and plasma sample from a volunteer 2.0 h after oral administration of SBT are compared and shown in Fig. 2. All the analytes and the IS were eluted within 11 min with retention time of 8.60, 6.65, 6.48, 8.98, 7.41, 2.03, 8.61, 7.56, 8.00 and 10.34 min for berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline, acetylcorynoline and the IS, respectively. No significant interfering peaks from endogenous substances were observed in the blank human plasma. 3.4.2. Linearity and sensitivity The typical regression equations of the calibration curves, which were calculated by a weighted (1/x2 ) least-squares linear regression analysis, are presented in Table 2. The correlation coefficient (r) for every calibration curve of the nine analytes was higher than 0.99. The LLOQs were 15.16 pg/mL for berberine, 15.93 pg/mL for epiberberine, 16.52 pg/mL for coptisine, 1.451 pg/mL for palmatine, 1.594 pg/mL for jatrorrhizine, 14.75 pg/mL for magnoflorine, 15.18 pg/mL for berberrubine, 11.90 pg/mL for corynoline and 14.73 pg/mL for acetylcorynoline, respectively. The values of the signal-to-noise ratio of these analytes at their LLOQs were higher than 10 at least, and the corresponding precision and accuracy of LLOQ samples presented in Table 3 were all within recommended limits. 3.4.3. Accuracy and precision The accuracy and precision of this method for the determination of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline are summarized in Table 3. All the values of RSD and RE were no more than 15% for all the QC levels for all the analytes. 3.4.4. Extraction recovery, matrix effect and carryover effect It was proved that this method was reproducible by studying the extraction recovery and normalized matrix effect for all the alkaloids (Table 4). The mean recovery data of the extraction procedure determined from QC samples were more than 77.1% for all the analytes. The values of normalized matrix factors of QC samples at two concentrations ranged from 90.3% to 111.2% with RSD less than 11.6%. No significant peak was found at the same retention times of the analytes in the chromatograms of blank plasma samples injected after the ULOQ samples. 3.4.5. Stability Table 5 shows the stability of all the analytes in human plasma for 10 h at room temperature, 65 days at −20 ◦ C, three freeze–thaw cycles, and the stability of all the analytes in autosampler for 29 h after being extracted. The stability of residue at room temperature for 24 h and at −20 ◦ C for 10 days is also listed in Table 5. There was no notable discrepancy between the measured concentration and spiked concentration. 3.4.6. Dilution integrity As shown in Table 6, the intra-precision and accuracy of dilution test at two concentration levels (low and high) were within the acceptable criteria. The results indicated that epiberberine, coptisine, palmatine, berberrubine, corynoline and acetylcorynoline could be assayed reliably by 5-fold diluting with blank plasma

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when the drug concentration in plasma samples exceeded the linear ranges of standard curves. 3.5. Application The validated method was applied to evaluate the concentration-time profiles of the nine alkaloids in human plasma after a single oral administration of SBT. The mean plasma concentration-time profiles of all the alkaloids in human plasma are presented in Fig. 3. Table 7 shows the important pharmacokinetic parameters of the two volunteers for all targets. Except of berberine, the half-life data of the other eight alkaloids in human have not been published in the literatures so far. After a single oral dose administration of Shanghua Baihe tablets, the mean half-life of berberine in the two volunteers was about 31 h. This result is consistent with that reported in the previous study, in which Hua et al. [27] reported that the mean half-life of berberine in human were about 30 h after a single oral dose administration of berberine hydrochloride tablets. But Huang et al. [28] stated that the mean half-life of berberine in healthy volunteers following the single oral administration of two TCM formulations, Rhizoma Coptidis granules and Jiao-Tai-Wan, were about 3.9 h and 4.7 h, respectively. The main reason that resulted in such a short half-life of berberine proposed by Huang et al. [28] may be caused by the too short a plasma sampling time of 24 h designed in their study, which made them almost miss the terminal phase of berberine pharmacokinetic profile. This implies that they had not got the real eliminate half-life of berberine in human. The other reason resulted in the different half-life data for the same compound in different TCM formulations obtained from different studies may be the influence of the other co-existing compounds in its TCMs [29]. 4. Conclusions To investigate the pharmacokinetics of the alkaloids of SBT in human, a highly sensitive LC–MS/MS method was developed and validated with respect to specificity, sensitivity, accuracy, precision and reproducibility. To our knowledge, the method in this study was firstly developed for the simultaneous quantification of berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline in human plasma. The higher sensitivity made the analytical procedure suitable for the determination of these low concentration alkaloids in human plasma. Then, the proposed method was successfully applied to pharmacokinetic study of the nine alkaloids in human after oral administration of SBT. The pharmacokinetic profiles of the alkaloids evaluated with this method are important for clinical application of SBT. Conflict of interest No potential conflict of interest was declared. Acknowledgments This study was supported financially by the National Natural Science Foundation of China (Grant 81273482) and the Graduate Innovation Fund of Zhejiang Huahai Pharmaceuticals Co., Ltd. References [1] Diana V. Messadi, F. Younai, Aphthous ulcers, Dermatol. Ther. 23 (2010) 281–290. [2] S. Jurge, R. Kuffer, C. Scully, S.R. Porter, Recurrent aphthous stomatitis, Oral Dis. 12 (2006) 1–21.

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