1. H M Arif Ullah, Hye Jin Chung*
Simultaneous determination of six anthraquinones
in rat plasma by LC-MS/MS
H M Arif Ullah, Hye Jin Chung*
College of Pharmacy, Gyeongsang National University, Jinju-daero 501, Jinju-52828, Republic of Korea
Abstract
A simple, sensitive, and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the simultaneous determination of six major anthraquinones in Rumex acetosa extract including emodin, emodin-8-O-beta-D-glucoside, chrysophanol, chrysophanol-8-O-beta-D-glucoside, physcion and physcion-8-O-beta-D-glucoside in rat plasma using dexibuprofen as an internal standard (IS). The chromatographic separation was carried out on a biphenyl column (3.0 × 100 mm, 2.6 µm) with a gradient elution within a runtime of 20 min. The mobile phase consisted of 5 mM ammonium formate (pH 6) aqueous solution (A) and methanol (B). A tandem mass spectrometric detection was accomplished by multiple-reaction monitoring (MRM) using an electrospray ionization (ESI) source in negative ion mode. The transitions monitored for anthraquinones were m/z 269.1/225.1 for emodin, 431.1/269 for emodin-8-O-beta-D-glucoside, 253.1/225.1 for chrysophanol, 415/253.1 for chrysophanol-8-O-beta-D-glucoside, 283/240.1 for physcion, 445.1/283.1 for physcion-8-O-beta-D-glucoside and 205/161.2 for IS, respectively. Validation of the assay was implemented including specificity and linearity.
Keywords: Anthraquinones, LC-MS/MS, Rat plasma
Introduction
Methods
A number of pharmacological assays revealed that the anthraquinone derivatives possess many pharmacological activities including antifungal, antimicrobial, anticancer, antioxidant, and antihuman cytomegalovirous activity. In addition, it has been reported that emodin shows anti- ulcer and anti-inflammatory activities. Chrysophanol was found to have antimicrobial activity Physcion has potential activity against human cervical cancer. However, no analytical method has been established to simultaneously quantify emodin, emodin-8-0-β-D-glucoside, chrysophanol, chrysophanol-8-0-β-D-glucoside, physcion and physcion-8-0-β-D-glucoside within one chromatographic run. Therefore, a simple, sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) is needed to determine the anthraquinones in the biological matrix. The aim of the present study was to develop a simple, rapid and sensitive LC-MS/MS method to concurrently quantify emodin, emodin-8-0-β-D-glucoside, chrysophanol, chrysophanol-8-0-β-D-glucoside, physcion and physcion-8-0-β-D-glucoside in rat plasma.
LC-MS/MS conditions
Instrument
Agilent 1260 infinity binary HPLC system (Agilent Technologies Inc., Germany) Agilent 6460 triple-quadrupole mass spectrometer (Agilent Technologies Inc., Singapore).
HPLC column
2.6 μm Biphenyl 110‎ Å, 100 × 3.0 mm
Mobile phase
(A) 5 mM ammonium formate (pH6) aqueous solution and (B) Methanol
Flow rate
0.250 μL/min
Injection volume
5 μL
Ionization method
ESI (electrospray ionization)
Detection mode
Negative ion, multiple reaction monitoring (MRM) mode
Structure of anthraquinone compounds
Sample preparation
An aliquot of 50 μL rat plasma was taken
Added 100 μL of dexibuprofen (0.1 μg/ml) in acetonitrile
After vortexing ½ min
Emodin
Crysophanol
Physcion
Centrifuged at 10,000 g, for 10 min then Supernatant collected
9, 10 anthraquinone
Evaporated at 30°C in centrifugal vaccum concentrator(60 min)
Reconstituted 50 μL of initial mobile phase
After vortexing ½ min Centrifuged at 10,000 g, for 10 min
Emodin-8-O-β-D-glucoside
Crysophanol-8-O β -D-glucoside
Physcion-8-O- β -D-glucoside
5 μL were injected into the LC-MS/MS
Results
Table 2. Calibration curve for anthraquinone compounds
Table 1. Optimized multiple reaction monitoring parameters for the detection of analytes and internal
standards
Compounds
Precursor ion (m/z)
Product ion (m/z)
Fragmentor
Collision energy (CE)
Emodin
Emodin-8-0-β-D- glucoside
Chrysophanol
Chrysophanol-8-0-β-D-glucoside
Physcion
Physcion-8-0-β-D-glucoside
IS (Dexibuprofen)
269.1
431.1
253.1
415
283.1
445.1
205
225.1
269
225.2
240.1
161.1
145
150
148 89 95 75 20 24 26 13 19 5
Nominal Conc.
(ng/mL)
Calculated Conc. (ng/mL)
Mean SD
Accuracy (%)
Precision (%)
Chrysophanol 1 2 3 10
8.45
11.6
9.50
1.82
95.00
19.14 20
21.08
23.31
21.82
1.29
109.12
5.90 50
46.4
38.55
43.78
4.53
87.57
10.35
100
92.7
89.7
91.70
1.73
1.89
200
208.7
171.9
208.68
196.43
21.24
98.21
10.81
500
543
499.2
543.02
528.41
25.29
105.68
4.79
Nominal Conc.
(ng/mL)
Calculated Conc. (ng/mL)
Mean SD
Accuracy (%)
Precision (%)
Emodin 1 2 3 10
10.17
11.17
10.50
0.58
105.03
5.50 20
22.71 24
23.14
0.74
115.70
3.22 50
56.12
55.8
56.11
56.01
0.18
112.02
0.32
100
105.23
112.4
107.62
4.14
3.85
200
187.07
187.8
187.31
0.42
93.66
0.23
500
431.5
428.7
430.57
1.62
86.11
0.38
Nominal Conc.
(ng/mL)
Calculated Conc. (ng/mL)
Mean SD
Accuracy (%)
Precision (%)
Emodin-8-O-β-D-glucoside 1 2 3 10
10.05
11.93
10.68
1.09
106.77
10.17 20
21.2
22.62
21.67
0.82
108.37
3.78 50
54.3
55.6
54.73
0.75
109.47
1.37
100
103.6
111.1
106.10
4.33
4.08
200
191.6
206.34
196.51
8.51
98.26
500
397.1
422.33
405.51
14.57
81.10
3.59
Nominal Conc.
(ng/mL)
Calculated Conc. (ng/mL)
Mean SD
Accuracy (%)
Precision (%)
Chrysophanol-8-O-β-D-glucoside 1 2 3 10
10.6
11.05
10.59
10.75
0.26
107.47
2.44 20
21.12
20.73
20.99
0.23
104.95
1.07 50
53.24
49.52
52.00
2.15
104.00
4.13
100
101.5
107.6
103.53
3.52
3.40
200
201.76
198.54
200.69
1.86
100.34
0.93
500
462.52
468.1
464.38
3.22
92.88
0.69
Physcion-8-O- β -D-glucoside
Emodin-8-O-β-D-glucoside
Emodin
Emodin-8-O-β-D-glucoside
Crysophanol-8-O β -D-glucoside
Physcion
Physcion-8-O-β-D-glucoside
Emodin
Chrysophanol-8-O-β-D-glucoside
Nominal Conc.
(ng/mL)
Calculated Conc. (ng/mL)
Mean SD
Accuracy (%)
Precision (%)
Physcion-8-O-β-D-glucoside 1 2 3 10
10.3
11.07
10.56
0.44
105.57
4.21 20
19.7
19.27
19.56
0.25
97.78
1.27 50
52.55
52.2
52.43
0.20
104.87
0.39
100
102.16
106.6
103.64
2.56
2.47
200
207.6
206.28
207.64
207.17
0.77
103.59
0.37
500
493.05
494.59
493.04
493.56
0.89
98.71
0.18
Chrysophanol
Crysophanol
Dexibuprofen
Fig 1. Representative MRM chromatograms of six anthraquinones and IS in rat plasma
Conclusion
A simple, specific and robust LC-MS/MS method has been developed for the simultaneous analysis of emodin, emodin-8-0-beta-D-glucoside, chrysophanol, chrysophanol-8-0-beta-D- glucoside, physcion and physcion-8-0-beta-D-glucoside in rat plasma. Further full validation and pharmacokinetic studies are needed of those anthraquinone compounds from Rumex acetosa extract. This result would be helpful for further study on their bioactivity.