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.