MS

Accepted Manuscript Title: Study on degradation kinetics of 2-(2-hydroxypropanamido) benzoic acid in aqueous solutions and identification of its major degradation product by UHPLC/TOF–MS/MS Author: Qili Zhang Jiao Guan Rong Rong Yunli Zhao Zhiguo Yu PII: DOI: Reference:

S0731-7085(15)00245-9 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.012 PBA 10048

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

30-12-2014 6-4-2015 8-4-2015

Please cite this article as: Q. Zhang, J. Guan, R. Rong, Y. Zhao, Z. Yu, Study on degradation kinetics of 2-(2-hydroxypropanamido) benzoic acid in aqueous solutions and identification of its major degradation product by UHPLC/TOFndashMS/MS, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Study on degradation kinetics of 2-(2-hydroxypropanamido)

2

benzoic acid in aqueous solutions and identification of its

3

major degradation product by UHPLC/TOF–MS/MS

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4 Qili Zhanga, Jiao Guana,b, Rong Ronga, Yunli Zhaoa, Zhiguo Yua

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a

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School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenhe District, Shenyang, 110016, China

b

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School of Pharmacy, Jilin Medical College, Jilin Street 5, Fengman District, Jilin, 132013, China

Abstract

A RP-HPLC method was developed and validated for the degradation kinetic

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study

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anti-inflammatory drug, which would provide a basis for further studies on HPABA.

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2-(2-hydroxypropanamido)

benzoic

acid

(HPABA),

a

promising

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The effects of pH, temperature, buffer concentration and ionic strength on the degradation kinetics of HPABA were discussed. Experimental parameters such as degradation rate constants (k), activation energy (Ea), acid and alkali catalytic constant (kac, kal), shelf life (t1/2) and temperature coefficient (Q10) were calculated.

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The results indicated that degradation kinetics of HPABA followed zero-order

20

reaction kinetics; degradation rate constants (k) of HPABA at different pH values

21

demonstrated that HPABA was more stable in neutral and near neutral conditions; the 

Corresponding author. Tel.: +86 24 23986295; Fax: +86 24 23986295. E-mail address: [email protected] (Z. Yu). 1

Page 1 of 33

function of temperature on k obeyed the Arrhenius equation (r = 0.9933) and HPABA

2

was more stable at lower temperature; with the increase of ionic strength and buffer

3

concentration, the stability of HPABA was decreased. The major unknown

4

degradation product of HPABA was identified by UHPLC/TOF–MS/MS with positive

5

electrospray ionization. Results demonstrated that the hydrolysis product was the

6

primary degradation product of HPABA and it was deduced as anthranilic acid.

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Keywords: 2-(2-Hydroxypropanamido) benzoic acid; Degradation kinetics;

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Aqueous solution; RP- HPLC; UHPLC/TOF–MS/MS; Identification

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1. Introduction

Marine organisms is regarded as one of the potential sources for

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pharmacologically active compounds and a few products (or their analogs) have

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already appeared in market as therapeutic drugs, health food, cosmetics, agrichemicals

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and enzymes [1-3]. In recent years, a growing number of pharmacologically active compounds with anti-inflammatory [4-7], anticancer [8-10], anti-angiogenesis [11], antioxidant [12, 13], antiviral and antibiotic properties [14-17] have been isolated from primary or secondary metabolism of marine organisms.

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2-(2-Hydroxypropanamido) benzoic acid (HPABA, Fig. 1A) was separated from

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the fermentation broth of marine fungi, Penicillium chrysogenum, which was obtained

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from North China sea. Initial investigations demonstrate that HPABA possesses

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significant anti-inflammatory and antinociceptive effects on mice at dose of 100

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Page 2 of 33

mg/kg, but it exhibits no ulcerogenic effect like aspirin [18]. In addition, the results of

2

carrageenan-induced rat hind paw edema and cotton pellet-induced rat granuloma

3

reveal that HPABA has significant effect on both acute and chronic inflammation and

4

the anti-inflammatory mechanisms of HPABA may be correlated to the decrease of

5

the level of prostaglandin E2 (PEG2), malondialdehyde (MDA) and nitric oxide (NO)

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and increase of the activities of superoxide dismutase (SOD). To further research on

7

HPABA, an efficient chemical synthetic route was developed for the batch synthesis

8

of HPABA. In addition, GC and RP-HPLC methods were established for investigating

9

its residue solvents and related substances; the content determination of HPABA was

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analyzed by the RP-HPLC method [19]. Furthermore, a rapid, sensitive and high

11

throughput UHPLC–MS/MS method was developed and validated to assay the

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concentration of HPABA in rat plasma and results of pharmacokinetic study indicate

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that HPABA has linear pharmacokinetic properties after intragastric administration

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within the dosage range[0] of 25-100 mg/kg and the absolute bioavailability is above

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59.1% [20].

Chemical stability study is essential to drug[0]s, because it can provide useful

information that how the quality of drug substances and drug products change over time under the influence of various environmental factors [21]. In addition, the

19

formation of degradation products may cause toxic or unexpected pharmacological

20

effects in patients, therefore, the identification and qualification of degradation

21

products are essential. For the identification of degradation products, HPLC

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hyphenated techniques, especially UHPLC/TOF–MS/MS, combining the advantages

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of UHPLC (high resolution, high sensitivity and high speed separation) with the exact

2

mass measurement offered by TOF mass spectrometry [22], have been increasingly

3

applied for the identification of unknown compounds. This approach has exhibited its

4

merits in providing exact mass of precursor ion and its fragmentation ions and the

5

elemental composition of corresponding ions, which has made it an attractive

6

analytical technique to analyze known compounds and elucidate unknown compounds

7

in complex matrices at fairly low levels, even when reference standards are not

8

available [23, 24]. In the structure of HPABA, there is an amide group, which is a

9

vulnerable and unstable chemical group. Therefore, investigations on the stability of

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HPABA in aqueous solutions are necessary. Nevertheless, there is no report on

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systematic stability study of HPABA and identification of its degradation product.

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The objectives of this study were to analyze the degradation kinetics of HPABA

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in aqueous solutions under different conditions and identify its major degradation

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product. The results of this study would do great help to further research on HPABA.

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2. Experimental

2.1. Materials and reagents

HPABA (purity > 99%) and anthranilic acid (degradation product, purity >

19

98.0%) were synthesized in School of Pharmacy, Shenyang Pharmaceutical

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University (Shenyang, China). Glacial acetic acid of HPLC grade was purchased from

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Concord Technology CO., Ltd (Tianjin, China). Formic acid and KH2PO4 of HPLC

22

grade were supplied by Kermel Chemical Reagent Co., Ltd (Tianjin, China).

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Acetonitrile of HPLC grade was purchased from Fisher Scientific (Fair Lawn, NJ,

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USA). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA,

3

USA). All the other reagents were of analytical grade.

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2.2. HPLC conditions

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Samples were analyzed on an HPLC system equipped with a Shimadzu LC-10AT

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pump, an AT-330 column oven and a SPD-10A detector (Kyoto, Japan).

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Chromatographic separation was performed on a CAPCELL PAK C18 column (150

9

mm × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile-water at a ratio of

10

20:80 (v/v) and the pH was adjusted to pH 3.0 with glacial acetic acid. The mobile

11

phase was delivered at a flow rate of 1 ml/min and ultraviolet detector was operated at

12

250 nm. The temperature of column was maintained at 35°C and the injection volume

13

was 20 µl.

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2.3. Method validation

The validation of the method was carried out according to ICH guidelines with

respect to specificity, linearity and range, precision and accuracy [25]. Specificity was evaluated by comparing chromatograms of blank phosphate

19

buffer spiked with the standard solution of HPABA with the degraded sample. To

20

evaluate linearity, a series of standard solutions (0.5, 2, 5, 20, 75, 100 and 150 µg/ml)

21

were prepared in phosphate buffer and assayed in duplicate. To determine the

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accuracy of method, three samples of HPABA at low, medium and high levels (1, 20

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Page 5 of 33

and 100 µg/ml) were analyzed on the same day and the accuracy was expressed as

2

recovery. The precision, represented as the relative standard deviation (RSD), was

3

examined by six analysis of HPABA with the interval in one day. Standard solution of

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20 µg/ml was determined in replicates (n = 6) on the same day to determine the

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repeatability of method.

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2.4. Degradation kinetic study

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2.4.1. The influence of pH on stability of HPABA

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HPABA solutions (200 µg/ml) were prepared using phosphate buffers of

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different pH values of 2.0, 6.5, 7.0, 8.0 and 9.0. The sample solutions were transferred

11

into glass tubes and then incubated at 70 °C in a water bath. According to a specified

12

time schedule, 100 µl of each HPABA solution was removed into EP tubes at 0, 0.25,

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0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12, 24, 36 and 48 h and then frozen at -20 °C until analysis,

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generally within 24 h. Before analysis, samples were neutralized to pH 7.0 with 100

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µl of hydrochloric acid or sodium hydroxide of appropriate concentrations and vortexed. The injection volume was 20 µl and each pH value was performed in duplicate.

2.4.2. The influence of temperature (T) on stability of HPABA

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HPABA solutions of 200 µg/ml were prepared in phosphate buffers at pH 8.0 and

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then incubated at 25, 37, 70 and 90 °C in a water bath. Other operations were similar

22

to 2.4.1.

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Page 6 of 33

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2.4.3. The influence of buffer concentration on stability of HPABA The impact of buffer concentration on stability of HPABA was examined under

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the condition of pH 8.0 and buffer concentration of 0.05, 0.1, 0.2 mol/L at 70 °C.

5

Other operations were similar to 2.4.1.

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2.4.4. The influence of ionic strength (µ) on stability of HPABA

The phosphate buffers (pH 8.0) with ionic strength of 0, 0.1, 0.3 and 0.5 at 70 °C

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were prepared to study the effect of ionic strength (µ) on stability of HPABA and

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sodium chloride was used to adjust ionic strength. Other operations were similar to

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2.4.1.

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2.5. Identification of major degradation product The major degradation product was identified by UHPLC/TOF-MS/MS and the

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analysis conditions as follows:

UHPLC/TOF-MS/MS analyses were carried out on a Waters Xevo G2 Q-TOF

mass spectrometer (Waters Corporation, Manchester, UK) equipped with an AcquityTM UPLC system (Waters Corporation, Milford, USA), quaternary pump,

19

vacuum degasser, autosampler and diode array detector. Chromatographic separation

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was conducted on a Thermo Syncronis C18 column (50 mm × 2.1 mm, 1.7 µm;

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Thermo, USA). The mobile phase was composed of acetonitrile – 0.1% formic acid in

22

water (20:80, v/v), which was delivered at 0.2 ml/min. The temperature of column and

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Page 7 of 33

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autosampler were maintained at 35◦C and 4◦C, respectively. The injection volume was

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2 l. Mass spectrometric detection was carried out using ESI source operating in

4

positive ion mode. The parameters of the mass spectrometer under the ESI mode were

5

as follows: ion source temperature 150 ◦C, cone gas flow 110 L/h, desolvation gas

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temperature 450 ◦C, desolvation gas flow 750 L/h, capillary voltage 3.0 kV and cone

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voltage 30 V. Full-scan MS data were collected from 50 to 1000 Da with collision

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energy of 20-40 eV and scan time of 0.2 s.

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Information about the accurate molecular weight was provided by TOF-MS

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spectrum, while the analyte could be identified with the help of characteristic

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fragmentation pattern provided by MS/MS spectra.

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3. Results and discussion

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3.1. Optimization of HPLC conditions

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The composition of mobile phase is a critical factor for the separation of HPABA

and other degradation products. In order to achieve satisfied chromatographic behavior, such as appropriate analysis time[0], good peak symmetry and separation, the mobile phase was optimized. Acetonitrile was chosen as the organic phase because

19

it provided higher responses and lower background noise by comparison with

20

methanol. Moreover, the ratio of organic component to aqueous in mobile phase was

21

also tested and a ratio of 20:80 (v/v) was selected. However, when acetonitrile–water

22

was chosen as the mobile phase, poor peak shapes with severe tailing were observed,

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Page 8 of 33

which might be caused by the residual ‘free silanol’ groups in the silica based reversed

2

phase stationary phase, which occurred mainly due to incomplete endcapping.

3

Therefore, the introduction of tailing-suppressing reagent into mobile phase was

4

necessary. Different acids including formic acid, glacial acetic acid and phosphoric

5

acid were evaluated in order to improve the peak symmetry of HPABA. It was found

6

that using glacial acetic acid in mobile phase was sufficient to achieve satisfied peak

7

shapes. Then, the pH of mobile phase was also investigated. The peaks were sharper

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and more symmetric when the pH value of mobile phase was adjusted to 3.0. The

9

underlying reason might be that the ionization of the weak acidic group and carboxyl

10

group was suppressed. Finally, a mobile phase consisting of acetonitrile-water at a

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ratio of 20:80 (v/v), which pH was adjusted to pH 3.0 with glacial acetic acid was

12

therefore chosen.

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3.2. Optimization of UHPLC/TOF-MS/MS conditions

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Acetonitrile was chosen as the organic phase because it could provide higher

responses and lower background noise by comparison with methanol. When adding formic acid to the mobile phase, the sensitivity and peak symmetry of HPABA and degradation product were remarkably improved. Different concentrations of formic

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acid were tested from 0.01 to 0.2% and addition of 0.1% formic acid was represented

20

to be with enough significant sensitivity for both analytes. Finally acetonitrile-0.1%

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formic acid in water (20:80, v/v) was chosen to be the mobile phase.

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Positive and negative ion modes were investigated in order to obtain the most

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Page 9 of 33

sensitive ionization method for analytes. The results showed that higher signal

2

intensity and more fragmentation ion information on the structures were obtained of

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HPABA and degradation product in the positive mode. Parameters including

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declustering potential (DP), collision energy (CE), entrance potential (EP) and cell

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exit potential (CXP) were optimized in order to achieve efficient separation and good

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responses to all chemical components.

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3.3. Method validation

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The peak shapes were good under the described conditions with no interference

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from other degradation products. The typical chromatograms of HPABA and degraded

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sample are shown in Fig. 2. Good linear relationship was established for HPABA in

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the investigated concentration range from 0.5 to 150 µg/ml. The typical calibration

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curve was y = 3.98 × 104 x + 1.1 × 104 (r = 0.9997), where y and x were the peak area

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and the concentration of HPABA, respectively. The results (listed in Table 1) of

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method validation indicated that the method had high accuracy, good precision and repeatability.

3.4. Degradation kinetic study

3.4.1. Determination of rate constant (k) and Shelf life (t1/2)

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The linear relationship between concentration and time and the regression

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coefficients (r) obtained indicated that the degradation of HPABA followed a

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zero-order rate kinetic. The observed zero-order rate constants (k) were calculated

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from the slopes of the concentration versus time in accordance with the following

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Page 10 of 33

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equation:

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C  C 0  kt

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Where C0 is the initial concentration and C is the remaining concentration of HPABA

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at time t.

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The regression equation, correlation coefficient (r), rate constant (k) and shelf life (t1/2) are shown in Table 2.

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t1 / 2  C 0 / 2 k

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The shelf life (t1/2) of the test drug was calculated as:

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(1)

3.4.2. Effect of pH values

As shown in Table 2, degradation rate constant (k) of HPABA at pH 6.5 was the

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smallest. So the maximum stability of HPABA was at pH 6.5. At pH below 6.5, the

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specific hydrogen-ion catalysis was observed. When the pH value is lower or higher

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than 6.5, the degradation rate constant was increased and the rate constants under alkaline conditions were larger than that under acidic conditions. The results showed a pH-dependent degradation of HPABA: in neutral and near neutral conditions, the degradation of HPABA was slow; while the pH increased or decreased, the stability of HPABA decreased.

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The acid catalytic constant (kac) and alkali catalytic constant (kal) were also

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calculated. Hydrolysis of drugs-containing amide bond was often catalyzed by H+ or

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OH- and the influence of pH on the k could be described by the following equation

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Page 11 of 33

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[26]:

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k  k 0  k ac [H  ]  k al [OH  ]

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where the k0 represents rate constant of water molecules involved in the catalytic

4

reactions, kac and kal are the acid catalytic constant and alkali catalytic constant,

5

respectively.

cr

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(3)

be expressed to:

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log k  log k ac  pH

(4)

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At a lower pH value, it is mainly acid-catalyzed reaction, the equation above can

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At a higher pH value, it is mainly alkali-catalyzed reaction, equation (2) can be

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expressed to:

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log k  log k al  log k w  pH , ( k w  [H  ][OH  ] )

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(5)

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Calculated by the above formula, kac and kal of HPABA at phosphate buffer (70

12 °

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than kac, which showed that the degradation of HPABA is mainly affected by OH- and

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C) were 106.3 h-1 and 10715 h-1, respectively. Under this condition, kal is much larger

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HPABA is more stable in the neutral condition.

3.4.3. Effect of temperature (T) The influence of temperature on the degradation of HPABA followed Arrhenius

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equation (Eq. (5)) [27]:

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ln k  ln A 

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where A and T are Arrhenius factor and the absolute temperature (K), respectively. Ea

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is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/k/mol) and k

Ea RT

(6)

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Page 12 of 33

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is degradation rate constant. Temperature coefficient (Q10) indicates the proportion increased of reaction rate

3

when temperature is increased by every 10 °C and calculated as the following

4

equation [28]:

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Q10  (

6

where k1 and k2 are the degradation rate constant of T1 and T2, respectively.

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k1 T ) k2

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(7)

The natural logarithm of degradation rate constant (lnk) of HPABA was linear

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with the reciprocal of absolute temperature. The typical calibration curve was lnk =

9

-1.08×1031/T + 3.13 (r = 0.9933). The regression equations could be used to

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calculated parameters of HPABA at any temperature in the same solutions. Especially,

11

the results obtained at 37 °C are important for future studies in vivo. The calculated k,

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t1/2, Ea and Q10 are shown in Table 3. The results showed that with the rise of T, t1/2 of

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HPABA was decreased and k was increased, which indicated that degradation of

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HPABA was accelerated by the increase of temperature. It is found that Q10 in high temperature range is larger than that in low temperature range, which explained that HPABA is not stable in a high temperature.

3.4.4. Effect of buffer concentrations

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Plotting the degradation rate constants (k) for HPABA in buffer solutions of

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different concentration versus the corresponding concentration (c) and a good

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linearity was obtained. As shown in Fig. 3, an increase of k for HPABA was observed

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as the buffer concentration increased, which suggests that phosphate buffer had an 13

Page 13 of 33

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effect on the hydrolysis of HPABA at pH 8.0. Thus, the stability of HPABA rose with

2

the concentration of buffer solution increasing.

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3.4.5. Effect of ionic strength (µ)

The influence of ionic strength on stability of drugs could be described by the

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following equation [29]:

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log k  log k 0  1.022Z A Z B  1 / 2

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where k represents the rate constant, k0 is the rate constant when ionic strength is zero,

9

ZA and ZB are the charges carried by reactants A and B, respectively.

(8)

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The influence of µ on the degradation of HPABA was evaluated by the plot of

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logk versus µ½. It is indicated that with the rise of ionic strength, the k value of

12

HPABA increased (Fig. 4). The underlying reason might be that salt reduce the

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electrostatic interactions among drugs. In brief, HPABA is more stable in low ionic

14

strength.

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3.5. Identification of major degradation product As illustrated in Fig. 2, one major degradation product was observed under

different conditions. The degradation product was observed at a retention time of 1.79

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min by UHPLC/TOF-MS/MS method with a typical [M+H]+ ion at m/z 138.0547,

20

suggested that its molecular formula was C7H7NO2 , which is consistent with

21

anthranilic acid (MW= 137). As shown in Fig. 5, the retention time of degradation

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product is consistent with that of anthranilic acid. In addition, the MS2 spectra of

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Page 14 of 33

major degradation product exhibited a series of product ions at m/z 120, 92, 77 and 65,

2

corresponded to that of anthranilic acid (Fig. 6). The predominant fragment ion at m/z

3

120 originated from the neutral loss of one water molecule. The ion at m/z 92 is

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generated by the removal of CO (28 Da) molecule from the ion at m/z 120. The ions at

5

m/z 77 and 65 are the characteristic fragment ions of benzene ring.

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From the above spectral information, the degradation product could be

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unambiguously confirmed as anthranilic acid (Fig. 1B), which was produced by the

8

hydrolysis of amide bond in the structure of HPABA. The proposed ESI–MS/MS

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fragmentation pathways of the degradation product are shown in Fig. 7.

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4. Conclusion

In this study, the established and validated RP-HPLC method was used for

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determination of HPABA and research on degradation kinetics of HPABA in aqueous

14

solution. It is indicated that degradation behavior of HPABA followed the zero-order

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kinetics; within the investigated pH values of 2-9, HPABA is most stable at pH 6.5; temperature can also affect the degradation of HPABA and a lower temperature makes it stable; with the increase of the concentration of buffer solution, the stability of HPABA was decreased; HPABA is more stable in a solution with lower ionic strength.

19

The

major

degradation

product

is

characterized

as

anthranilic

acid

by

20

UHPLC/TOF–MS/MS. The information of degradation kinetics will be useful for

21

understanding the chemical stability of HPABA. Therefore, in development of

22

suitable formulations and proper storage conditions of HPABA, the influence of pH,

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Page 15 of 33

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temperature, buffer concentration and ionic strength on the stability of it should be

2

taken into account and these factors should be controlled properly.

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This work was partly financed by the National Innovation Drugs in Liaoning

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Acknowledgement

Province (Grant No. 2009ZX09301-012).

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[2] D.J. Faulkner, Marine natural products, Nat. Prod. Rep. 19 (2002) 1–49.

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research on marine natural products based on data obtained between1985 and 2008, Mar. Drugs

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[4] C. Festa, S. De Marino, V. Sepe, M.C. Monti, P. Luciano, M.V. D’Auria, C. Débitus, M.

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Bucci, V. Vellecco, A. Zampella, Perthamides C and D, two new potentanti-inflammatory

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cyclopeptides from a Solomon Lithistid sponge Theonella swinhoei, Tetrahedron 65 (2009)

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[5] B.W. Chen, C.H. Chao, J.H. Su, Z.H. Wen, P.J. Sung, J.H. Sheu, Anti-inflammatory eunicellin-based diterpenoids from the cultured soft coral Klyxum simplex, Org. Biomol, Chem. 8 (2010) 2363–2366.

[6] W.Y. Lin, J.H. Su, Y. Lu, Z.H. Wen, C.F. Dai, Y.H. Kuo, J.H. Sheu, Cytotoxic and

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Bioorg. Med. Chem. 18 (2010) 1936–1941

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2

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Nitrogenous sesquiterpenes from the Thai marine sponge Halichondria sp, Tetrahedron 67 (2011)

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Linley, D. Divlianska, J.K. Reed, A.E. Wright, Leiodermatolide, a potent antimitotic macrolide

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Lagunamide C, a cytotoxic cyclodepsipeptide from the marine cyanobacterium Lyngbya

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majuscula, Phytochemistry 72 (2011) 2369–2375.

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angiogenesis inhibitor from a marine-derived Streptomyces sp. KORDI-3973, Phytochemistry

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chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2, Carbohydr. Polym. 78 (2009) 117–124. [13] K. Schneider, J. Nachtigall, A. Hanchen, G. Nicholson, M. Goodfellow, R.D. Sussmuth, H.P. Fiedler, Lipocarbazoles, secondary metabolites from Tsukamurella pseudospumae acta 1857 with

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antioxidative activity, J. Nat. Prod. 72 (2009) 1768–1772.

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[14] O. Bergman, B. Mayzel, M.A. Anderson, M. Shpigel, R.T. Hill, M. Ilan, Examination of

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marine-based cultivation of three demosponges for acquiring bioactive marine natural products,

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Mar. Drugs 9 (2011b) 2201–19.

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Additional bioactive guanidine alkaloids from the Mediterranean spongeCrambe crambe, RSC

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Adv 2 (2012) 2828–35.

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advanced preclinical trials, J. Nat. Prod. 67 (2004) 1216–38.

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Mar. Biotechnol. 7 (2005 a) 142–62.

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metabolites of the marine fungus Penicillium chrysogenum, Chem. Nat. Compd. 50 (2014)

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of the related substances of 2-(2-hydroxypropanamido) benzoic acid by RP-HPLC, J. Shenyang

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Pharm. Univ. 31 (2014) 21–25.

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[20] J. Guan, Y.L. Zhao, H.Y. Zhu, Z.Z. An, Y.M. Yu, R.J. Li, Z.G. Yu, A rapid and sensitive

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UHPL-MS/MS method for quantification of 2-(2-hydroxypropanamido) benzoic acid in rat plasma: Application to a pharmacokinetic study, J. Pharm. Biomed. Anal. 95 (2014) 20–25. [21] R. Li, F.Z. Wang, L. Chen, S.N. Zhu, L. Wu, S.M. Jiang, Q.W. Xu, D.Y. Zhu, Stability of an anti-stroke peptide: Driving forces and kinetics in chemical degradation. Int. J. Pharm. 472 (2014)

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Characterization and identification of steroidal saponins from the seeds of Trigonella

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foenum-graecum by ultra high-performance liquid chromatography and hybrid time-of-flight

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13

Pharm. Univ. 25 (2008) 615-619.

14

[27] J.O. Fubara, R.E. Notari, Influence of pH, temperature and buffers on cefepime degradation

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kinetics and stability predictions in aqueous solutions, J. Pharm. Sci. 87 (1998) 1572–1576. [28] X. D. Cao, X. G. Fang, K. Zhao, Z. L. Yao, F. F. Li, Degradation kinetics and effects of ascorbic acid on thermal stability of anthocyanins in blueberry (Vaccinium ashei) juice, J. Chin.

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19

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20

aqueous systems, Int. J. Pharm. 259 (2003) 129–141.

21 22

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Page 20 of 33

1 2

Figure Captions:

4

Fig. 1. Chemical structures of HPABA (A) and its major degradation product (B).

5

Fig. 2.The chromatograms of the blank phosphate buffer added with the standard solution of

6

HPABA (A), the degraded sample under alkaline condition (B) and acidic condition (C): a is the

7

major degradation product.

8

Fig. 3. The degradation rate of HPABA over different buffer concentrations at pH 8.0 and 70 C.

9

Fig. 4. Influence of ionic strength on the stability of HPABA at pH 8.0 and 70 C.

an

us

cr

ip t

3

°

°

Fig. 5. Representative chromatograms of the degraded sample (A) and anthranilic acid (B): 1

11

degradation product, 2 anthranilic acid.

12

Fig. 6. Positive ion electrospray ionization mass spectra for anthranilic acid reference standard (a)

13

and degradation product (b).

14

Fig. 7. The proposed ESI–MS/MS fragmentation pathways of the major degradation product.

16 17 18

d

te

Ac ce p

15

M

10

19 20 21 22 23 21

Page 21 of 33

（A）

cr

ip t

1 2

(B)

an

4 5 6

us

3

8 9 10 11 12

Ac ce p

te

d

M

7

13 14

Fig. 1.

15 16 22

Page 22 of 33

Itens.(mv)

100

A

HPABA

90 80 70 60

ip t

50 40 30 20

0 0

1

2

3

4

5

6

7

8

T(min)

us

100

B

HPABA

90 80 70 60

M

50 40 30 20

d

10 0

2

1

2

3

4

a 5

6

7

100

8

9

T(min)

Ac ce p

te

0

Intens.(mv)

9

an

Intens.(mv)

1

cr

10

C

HPABA

90 80 70 60 50 40 30 20 10

a

0 0

3 4 5 6 7

1

2

3

4

5

6

7

8

9

T(min)

Fig. 2.

8 23

Page 23 of 33

1.5 1

ip t

k (µg/ml/h)

2

0 0.05

0.1

0.15

C (mol/L)

1

M d te Ac ce p

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

0.25

an

2 3

0.2

us

0

cr

0.5

Fig. 3.

31 24

Page 24 of 33

1

0.4

0 -0.2

0

0.2

0.4

0.6

1/2

us

µ

Ac ce p

te

d

M

an

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

0.8

cr

-0.4

ip t

logk

0.2

Fig. 4.

31

25

Page 25 of 33

us

cr

ip t

1

an

2 3

5 6 7 8 9 10 11 12 13 14

Ac ce p

te

d

M

4

Fig. 5.

15

26

Page 26 of 33

cr

ip t

1

6 7 8 9 10 11 12 13

te

5

Ac ce p

3 4

d

M

an

us

2

14 15 16 17 18

Fig. 6.

19

27

Page 27 of 33

us an M d te Ac ce p

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

cr

ip t

1 2

Fig. 7.

36 28

Page 28 of 33

ip t cr us an M d te Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Graphical abstract

25 29

Page 29 of 33

1

Highlights

2

1. Development of a RP-HPLC method to study the degradation kinetics of HPABA.

4

2. In this study, the various influence factors were considered.

5

3. A LC/TOF–MS/MS method was developed to identify the degradation product.

6

4. The degradation rate obtained indicated a zero-order reaction law.

7

5. Results of this study would do great help to further research on HPABA.

cr

ip t

3

Ac ce p

te

d

M

an

us

8

30

Page 30 of 33

1

Table 1.The results of the method validation.

Concentration Recovery

RSD

99.6%

20

100.2%

100

99.2%

20

Repeatability

20

0.48% 0.80%

us

Precision

2

an

3

M

4 5

9 10 11 12 13

te Ac ce p

8

d

6 7

0.51%

cr

Accuracy

1

ip t

(µg/ml)

14 15 16 17 31

Page 31 of 33

°

1

Table 2. Regression equations of degradation rate for different pH values at 70 C and the

2

corresponding rate constant (k) and shelf life (t1/2).

8 9 10 11

t1/2(h)

0.9969

1.0633

6.5

C = -0.9559 t + 86.088

0.9956

0.9559

7.0

C = -1.0278 t + 85.294

0.9951

8.0

C = -1.5787 t + 86.194

0.9956

9.0

C = -1.6956 t + 87.206

us

an

46.95

1.0278

43.39

1.5787

28.66

1.6956

26.89

M

0.9980

41.97

cr

C = -1.0633 t + 84.96

d te Ac ce p

7

(µg/ml/h)

2.0

4

6

coefficient(r)

Regression equation

3

5

k

ip t

pH

Correlation

12 13 14 15

32

Page 32 of 33

1

Table 3. The calculated values of rate constant (k), shelf life (t1/2), activation energy (Ea) and

2

temperature coefficient (Q10 ).

k(µg/ml/h)

t1/2(h)

25

0.3075

146.29

Ea(kJ/mol)

Q10

ip t

T(°C)

25-37°C

0.5112

88.00

cr

37

8.943

90

0.9559

47.06

1.4722

30.56

4

10 11 12 13

1.241

te Ac ce p

9

70-90°C

d

6

8

1.209

M

5

7

37-70°C

an

3

us

70

1.527

14 15 16

33

Page 33 of 33