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. 2017 Dec 5;7:16921. doi: 10.1038/s41598-017-17170-y

Chemical profiling and quantification of XueBiJing injection, a systematic quality control strategy using UHPLC-Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry

Zhi Sun 1, Lihua Zuo 1,✉, Tongwen Sun 2, Jinfa Tang 4, Daling Ding 3, Lin Zhou 1, Jian Kang 1, Xiaojian Zhang 1,✉
PMCID: PMC5717239  PMID: 29208914

Abstract

To clarify and quantify the chemical profiling of XueBiJing injection (XBJ) rapidly, a feasible and accurate strategy was developed by applying ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). A total of 162 components were characterized, including 19 phenanthrenequinones, 33 lactones, 28 flavonoids and 12 phenolic acids and 51 other compounds. Among them, 38 major compounds were unambiguously quantified by comparing with reference standards. Meanwhile, 38 representative compounds were simultaneously detected in XBJ samples by Q-Orbitrap HRMS. Satisfactory linearity and correlation coefficient were achieved with wide linear range. The precisions, repeatability, stability and recovery were meeting requirements. The validated method was successfully applied for simultaneous determination of 38 bioactive compounds in 10 batches XBJ samples. In addition, the similarity evaluation of fingerprintings was applied to assess the quality of XBJ. And the results were evaluated by multiple statistical strategies and five compounds might be the most important chemical markers for chemical quality control of XBJ. Finally, a rapid and simple UPLC-MS/MS method was developed for determination of five markers in XBJ sample. This research established a high sensitive and efficient strategy for integrating quality control, including identification and quantification of XBJ.

Introduction

XueBiJing injection (XBJ) was comprised of extracts from five Chinese herbals: Carthami Flos, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Salviae miltiorrhizae and Angelicae Sinensis Radix. It has been widely used in China as a blood-activating and anti-endotoxicity drug for the treatment of sepsis and the associated multiple organ dysfunction syndrome (MODS)1,2. Modern pharmacological studies indicate that XBJ could protect the endothelium, improve microcirculation, alleviate coagulation and inflammation, and regulate immune response3,4. In clinical, XBJ could significantly reduce significantly the value of serum procalcitonin, C-reactive protein and the level of white blood cells in sepsis patients. In addition, the XBJ had an antagonistic effect on inflammatory markers, which could interdict the pathological process of systemic inflammatory response syndrome and reduce the incidence of MODS in order to further improve the prognosis of sepsis patients and reduce the mortality5,6. Although XBJ is an effective traditional Chinese medicine (TCM) in treating sepsis, the constituents of which remain largely unknown, and the bioactive components are not completely clear.

According to previous phytochemical and HPLC or UPLC-MS researches, glycosides, flavonoids and phenolic acids were the predominant constituents in XBJ. To date, a few reports have developed a method for qualitative or/and quantitative analysis of compounds in XBJ7–10. Ji et al.7 established a HPLC method coupled with an ultraviolet detector for the determination of 11 essential compounds in XBJ within 70 min, deficiency existed in terms of analysis time and sensitivity. Huang et al.9 developed an ultra performance liquid chromatographic (UPLC) method for simultaneous identification and quantification of 13 main components in XBJ and an UPLC/Q-TOF method for identification of 8 major metabolites in XBJ. Huang et al.11 established an HPLC/DAD/TOF method to identify 23 compounds in XBJ, including amino acids, phenolic acids, flavonoid glycosides, terpene glycosides and phthalides. However, due to the limitation of applied instruments, only high level components were studied in previous studies. To develop a sensitive and accurate method for the comprehensive chemical identification of XBJ, Q-Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometer (Q-Orbitrap HRMS) was employed in the present study.

In this paper, qualitative and quantitative analyses were combined together for the integrated quality control strategy of XBJ. In qualitative analysis, Q orbitrap MS revealed its remarkable high resolution and sensitivity in the chemical identification of XBJ. Q-orbitrap HRMS was employed in the analysis of Chinese medicinal formula for the first time, and it overcame the drawbacks of HPLC and UPLC-MS. In present investigation, 162 unknown compounds were identified, based on their high resolution MS data and the cleavage patterns of 38 reference standards. Meanwhile, in order to avoid the ion response discrimination to different types constituents in XBJ, the fast polarity swinging was realized in one analysis. In addition, the utilization of Q orbitrap HRMS could realize simultaneously qualitative and quantitative determination in one analysis, which shortened analysis time. To the best of our knowledge, this is the first time to report the application of Q-Orbitrap HRMS in simultaneously determining and quantifying so many bioactive constituents in XBJ. The quantitative determination method had been validated and applied for an assay of 10 bathes XBJ samples, and the result could evaluated by fingerprinting and multivariate data analyses (principal component analysis, PCA). Finally, a rapid and simple UPLC-MS/MS method was developed for determination of five markers in XBJ. In one word, we provided a promising and integrated approach for the quality control of XBJ and a solid foundation for the pharmacological and pharmacokinetic study of XBJ.

Results and Discussions

Qualitative analysis of XBJ

A specific UHPLC-Q-Orbitrap HRMS method was developed as a reliable, sensitive and high-throughput method for rapid identification of the components of XBJ regardless of the macro- and micro-constituents. The total ion chromatograms (TIC) of the XBJsample both in positive and negative ion mode are presented in Fig. 1 38 compounds were unambiguously identified based on comparison of retention time and high-resolution accurate mass with that of available reference standards and their chemical structures were shown in Fig. 2. Moreover, the fragmentation patterns and pathways of the standards were investigated in depth to further confirm the structure of their derivatives. For the compounds without available references, the structures were presumed based on the following steps so as to increase the credibility: (1) the molecular formula was established based on high-accuracy protonated precursors such as [M + H]+, [M + Na]+, [M−H]−, or [M + HCOO]− within a mass error of 10 ppm and the fractional isotope abundance; (2) A class of compounds has the same law of cracking, therefore, the standards were utilized to characterize the fragment pathways and diagnostic fragment ions that could be applied for structural elucidation of their derivatives. In addition, some literatures about the compositions of XBJ and five Chinese herbals could be referred. (3) The fragment ions from mass spectrometry were used to further confirm the chemical structure with the aid of Thermo ScientificTM Mass Frontier 7.012.

Figure 1.

Figure 1

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The total ion chromatograms (TIC) of the XBJ sample (A) in positive mode (B) in negative mode.

Figure 2.

Figure 2

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Chemical structures of 38 major components identified from XBJ injection.

As for monoterpene glycosides, the loss of CH3, H2O and CO was observed clearly in their MS/MS spectra. The mass spectra and proposed major fragmentation of representative compounds Paeoniflorin was shown in Fig. 3A and the proposed fragmentation pathways was presented in Fig. 3B. Other constituents were tentatively deduced by the above steps and paeonisuffrone, phenanthrenequinone, senkyunolide, lactones, flavonoids and phenolic compounds dominated the chemical profiling of XBJ13–23. Overall, 162 components, including 19 monoterpene glycosides, 19 phenanthrenequinone, 33 lactones, 28 flavonoids and 63 phenolic acid and other compounds, in XBJ were identified or tentatively characterized with their retention times and MS data, which are summarized in Table 1.

Figure 3.

Figure 3

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(A) The mass spectra and proposed major fragmentation of Paeoniflorin (B) Proposed fragmentation pathways of Paeoniflorin.

Table 1.

19 monoterpene glycosides (M), 19 phenanthrenequinone (P), 33 lactones (L), 28 flavonoids (F), 63 phenolic acid and other compounds (O) identified from XBJ by UHPLC-Q-Exactive.

No. Compounds t R (min) Formula Ion mode ES/expected (m/z) ES/measured (m/z) Delta (ppm) HPLC-ESI-MS/MS (m/z)
M1 1-O-β-D-glucopyranosyl-paeonisuffr-one 3.21 C16H24O9 − 359.13475 359.13400 −1.853 197.08099, 179.07028
M2 4-O-Methyldesbenzoylpaeoniflorin 8.12 C17H26O10 − 435.15080 435.14944 −3.032 389.14432, 227.09203
M3 Mudanpioside F 9.90 C16H24O8 − 343.13984 343.13879 −3.063 181.08582, 151.07512, 109.06439
M4 6′-O-galloyl-Desbenzoylpaeoniflorin Isomer 12.02 C23H28O14 − 527.14063 527.13922 −2.672 497.12943,479.11896,399.09393,313.05627,271.04553(C11H11O8),211.02382,169.01294
M5 6′-O-galloyl-Desbenzoylpaeoniflorin Isomer 16.34 C23H28O14 − 527.14063 527.13922 −2.672 491.11990,345.11871,313.05621,271.04556, 211.02365(C9H7O6), 169.01297
M6 Oxypaeoniflorin a (A12) 18.15 C23H28O12 − 495.15080 495.14963 −2.362 495.15009,333.09671,281.06604,195.06506, 165.05431, 151.03847, 137.02304
M7 6′-O-galloyl-Desbenzoylpaeoniflorin Isomer 20.27 C23H28O14 − 527.14063 527.13947 −2.198 497.13281,399.09381,313.05579,271.04590, 211.02440, 169.01299
M8 Albiflorin a (A14) 23.68 C23H28O11 − 479.15588 479.15445 −2.994 479.11319, 327.10904, 121.02808
M9 Paeoniflorin a (A15) 25.31 C23H28O11 − 479.15588 479.15524 −2.619 449.14481,367.11890,357.18002, 121.02803
M10 Oxypaeoniflorin isomer 26.65 C23H28O12 − 495.15080 495.14890 −1.816 465.13937, 165.05487, 137.02306
M11 Paeoniflorin Isomer 29.44 C23H28O11 − 479.15588 479.15454 −2.807 479., 327.10953, 165.05482, 121.02806
M12 Galloylpaeoniflorin isomer 31.48 C30H32O15 − 631.16684 631.16498 −2.952 631.16595,613.15570,491.11874,399.09253,313.05603,271.04556,211.02396,169.01303
M13 Paeoniflorin Isomer 31.63 C23H28O11 − 479.15588 479.15414 −2.497 479.15417, 327.11026, 263.07455, 177.05457, 165.05447, 121.02803
M14 Galloylpaeoniflorin isomer 32.94 C30H32O15 − 631.16684 631.16516 −2.667 491.12109, 399.09271, 313.05630, 271.04538, 211.02423, 169.01303, 121.02809
M15 Galloylpaeoniflorin isomer 33.44 C30H32O15 − 631.16684 631.16492 −3.047 491.12549, 399.09335, 313.05603, 271.04532, 211.02380, 169.01299, 121.02781
M16 Galloylpaeoniflorin isomer 34.71 C30H32O15 − 631.16684 631.16510 −2.762 431.12598, 313.05792, 169.01309, 121.02789
M17 Benzoyloxypaeoniflorin Isomer 39.58 C30H32O13 − 599.17701 599.17554 −2.460 477.13870,431.13376,281.06613,239.05521, 137.02303, 121.02802
M18 Benzoyloxypaeoniflorin Isomer 41.37 C30H32O13 − 599.17701 599.17566 −2.260 477.14032, 385.09171, 333.09769, 281.06573, 165.05461, 137.02309, 121.02803
M19 Benzoylpaeoniflorin a (A30) 47.43 C30H32O12 − 583.18210 583.18005 −0.953 481.16913,431.13596,165.05434, 135.04375, 121.02801
P1 Tanshinone VI 41.83 C18H16O4 + 297.11213 297.11133 −2.711 279.15570, 261.09055, 184.01868
P2 Cryptotanshinone isomer 50.35 C19H20O3 + 297.14852 297.14746 −3.571 253.15790, 238.13448
P3 Tanshinone IIB 50.92 C19H20O4 + 313.14344 313.14255 −2.924 313.14240, 295.13196, 269.15262, 251.14232
P4 Tanshinone IIB-isomer 51.09 C19H20O4 + 313.14344 313.14264 −2.541 313.14260, 295.13177, 277.12137, 251.14224
P5 Tanshinone IIB-isomer 52.45 C19H20O4 + 313.14344 313.14149 −3.020 313.14246, 295.13196, 285.14700, 267.13751
P6 Tanshinone V 53.06 C19H22O4 + 315.15909 315.15811 −3.191 315.15793, 297.14737, 279.13748, 267.13718
P7 Tanshinone I isomer 53.47 C18H12O3 + 277.08592 277.08533 −2.457 277.08524, 249.09021, 178.07695
P8 Miltiodiol 53.65 C19H22O3 + 299.16417 299.16432 −2.511 299.16278, 281.15341, 253.15788
P9 Deoxyneocryptotanshinone 53.65 C19H22O3 + 299.16417 299.16339 −2.611 299.16278, 281.15314, 253.15788
P10 1,2,5,6-tetrahydrotanshinone I 53.72 C18H16O3 + 281.11722 281.11658 −2.280 182.08078, 72.08125
P11 Tanshinaldehyde 54.19 C19H16O4 + 309.11213 309.11136 −2.315 309.11087, 291.09970, 265.12158,223.07478
P12 Tanshinone V-isomer 54.97 C19H22O4 + 315.15909 315.15823 −2.715 297.14822, 253.15796
P13 Cryptotanshinone isomer 54.83 C19H20O3 + 297.14852 297.14792 −0.601 297.14749, 253.15787
P14 Tanshinone IIB-isomer 55.24 C19H20O4 + 313.14344 313.14249 −3.020 313.14249, 295.13171, 269.15353, 251.14236
P15 TanshinoneαA isomer 55.25 C19H18O3 + 295.13287 295.13211 −2.578 295.13104, 267.13715, 184.01865
P16 Dihydrotanshinone I 55.45 C18H14O3 + 279.10157 279.15836 −2.600 279.09811, 167.03360, 149.02304
P17 Tanshinone I a (A35) 57.26 C18H12O3 + 277.08592 277.08524 −2.132 277.08493, 249.09039, 221.09573, 178.07707
P18 Cryptotanshinone a (A36) 57.32 C19H20O3 + 297.14852 297.14774 −2.628 279.13742,251.14249
P19 TanshinoneαA a(A38) 59.12 C19H18O3 + 295.13287 295.13245 −1.426 295.13208, 277.12158, 249.12682,20708025
L1 Senkyunolide J/N isomer 22.69 C12H18O4 + 227.12778 227.12724 −2.402 209.11659, 191.10608
L2 Senkyunolide I/H isomer 25.86 C12H16O4 + 225.11213 225.11154 −2.645 207.10107, 165.09067, 137.09589
L3 Senkyunolide I/H isomer 27.68 C12H16O4 + 225.11213 225.11166 −2.112 207.10107, 165.09065, 137.09589
L4 Senkyunolide J/N isomer 30.45 C12H18O4 + 227.12778 227.12712 −2.930 249.10902, 209.11671, 191.10614, 163.11134, 153.05424
L5 Senkyunolide J/N isomer 31.30 C12H18O4 + 227.12778 227.12721 −2.534 249.10927, 209.11664, 191.10611, 163.11128, 153.05421
L6 Perloyrine 32.40 C16H12N2O2 + 265.09715 265.09641 −2.807 247.08580, 219.09067, 206.08324, 185.07040
L7 Senkyunolide F isomer 32.90 C12H14O3 + 207.10157 207.10114 −2.080 189.09055, 161.09563
L8 Senkyunolide I/H isomer 32.92 C12H16O4 + 225.11213 225.11145 −3.045 247.09338, 207.10104, 189.09039, 165.09076
L9 Senkyunolide J/N isomer 33.13 C12H18O4 + 227.12778 227.12715 −2.798 209.11665, 191.10616, 163.11128, 153.05423
L10 Senkyunolide I/H a (A21) 34.94 C12H16O4 + 225.11213 225.11130 −3.712 247.09317, 207.10097, 189.09077, 165.09033
L11 Senkyunolide F isomer 34.97 C12H14O3 + 207.10157 207.10089 −3.228 207.10103, 189.09053, 161.09546
L12 Senkyunolide F isomer 36.89 C12H14O3 + 207.10157 207.10118 −1.887 207.10103, 189.09055, 161.09578
L13 SenkyunolideB/C/E isomer 41.50 C12H12O3 + 205.08592 205.08531 −2.978 187.07489, 177.09053, 163.03853, 149.02296
L14 E/Z-Butylidenephthalide 44.13 C12H12O2 + 189.09101 189.09052 −2.571 171.08003, 161.09569, 153.06956
L15 SenkyunolideB/C/E isomer 44.27 C12H12O3 + 205.08592 205.08542 −2.442 187.07477, 163.03853, 149.02298
L16 E/Z-Butylidenephthalide 50.04 C12H12O2 + 189.09101 189.09041 −3.153 171.07994, 153.06944, 133.02815
L17 Butylidenephthalide isomer 50.04 C12H12O2 + 189.09101 189.09041 −0.616 189.09045,171.07994,161.09569,143.08519,133.02815
L18 SenkyunolideG/K 50.31 C12H16O3 + 209.11722 209.11652 −3.352 173.09526,163.11130,149.05936,145.10080,135.04381
L19 Ligustilides isomer 50.31 C12H14O2 + 191.10666 191.10603 −0.636 191.10611, 149.05936, 135.04381
L20 Neocnidilide 50.59 C12H18O2 + 195.13796 195.13741 −2.800 177.12680, 167.14268, 159.11650, 81.07021
L21 Senkyunolide A isomer 50.77 C12H16O2 + 193.12231 193.12196 −1.793 193.12181, 175.11171, 165.12669, 149.02275, 137.05954,85.06510, 57.07050
L22 Senkyunolide A isomer 51.72 C12H16O2 + 193.12231 193.12199 −1.638 165.12685, 147.11612, 137.05928, 85.06510, 57.07080
L23 SenkyunolideB/C/E isomer 52.37 C12H12O3 + 205.08592 205.08553 −1.905 187.07495, 169.06419, 159.08002, 149.02292,
L24 Senkyunolide M isomer 52.57 C16H22O4 + 279.15908 279.15839 −2.492 301.14026;261.14850, 233.15289, 215.14252, 191.10616, 173.09566, 71.049963
L25 Senkyunolide M isomer 53.36 C16H22O4 + 279.15908 279.15842 −2.385 301.14023,261.14780,251.16348,243.13681,233.15300,191.10619, 149.02301, 71.04964
L26 Senkyunolide A 53.53 C12H16O2 + 193.12231 193.12212 −0.965 175.11130,147.11649, 137.05946,93.07011
L27 Senkyunolide M isomer 53.69 C16H22O4 + 279.15908 279.15836 −2.600 261.14764,233.15303,149.02307,105.03358,71.04965
L28 Senkyunolide A isomer 53.72 C12H16O2 + 193.12231 193.12192 −2.000 175.11131, 147.11650, 137.05945
L29 Ligustilide a (A33) 55.41 C12H14O2 + 191.10666 191.10637 −1.498 173.09579, 163.11143, 145.10085
L30 Butylidenephthalide a (A34) 55.51 C12H12O2 + 189.09101 189.09077 −1.249 171.08009,161.09583,153.06960, 149.02299, 133.02811
L31 Cnidumlactone B 55.74 C24H30O5 + 399.21660 399.21567 −2.331 421.19784, 307.16711, 191.10616
L32 Levistolide A isomer 55.76 C24H28O4 + 381.20603 381.20499 −2.744 335.15710, 307.16754, 251.10570, 191.0612
L33 Levistolide A a (A37) 58.88 C24H28O4 + 381.20603 381.20502 −2.665 381.20532, 363.19830, 191.10625
F1 Catechin a (A9) 16.02 C15H14O6 − 289.07176 289.07108 −0.415 289.07120,245.08130,221.08067,203.07079,179.03343,165.01825,151.03885,137.02296
F2 Quercetin-O-2glu/gal isomer 19.79 C27H30O17 + 627.15557 627.15369 −3.007 465.10092, 355.40744,303.04895, 127.03870, 85.02866, 69.03396
F3 Quercetin-O-2glu/gal isomer 21.69 C27H30O17 + 627.15557 627.15363 −3.102 465.10059, 303.04892, 127.03899, 85.02874
F4 Quercetin-O-2glu/gal isomer 22.34 C27H30O17 + 627.15557 627.15338 −3.501 465.10056, 303.04895, 127.03912, 85.02863
F5 Quercetin-O-2glu/gal isomer 26.42 C27H30O17 + 627.15557 627.15369 −3.007 465.10049,303.04901,287.05411,127.03870,85.02872
F6 Quercetin-O-2glu/gal isomer 26.78 C27H30O17 + 627.15557 627.15405 −2.433 627.15643,465.10114,303.04889,288.05804,177.05420,127.03889, 145.02821, 85.02873
F7 Rutin isomer 27.05 C27H30O16 + 611.16066 611.15894 −2.816 449.10614, 287.05414
F8 Kaempferol-O-Glc-isomer 28.86 C21H20O11 + 449.10784 449.10632 −3.380 287.05411;153.01807, 121.02850,85.02871
F9 Rutin isomer 28.86 C27H30O16 + 611.16066 611.15863 −3.323 287.05405, 145.04912, 85.02869
F10 Quercetin-isomer 29.66 C15H10O7 + 303.04993 303.04898 −3.132 285.03848, 275.01776, 257.01776
F11 Quercetin-isomer 30.31 C15H10O7 + 303.04993 303.04901 −3.033 303.01257,257.04388, 229.04872,165.01776,153.01802
F12 Rutin a (A17) 30.31 C27H30O16 + 611.16066 611.15912 −2.522 303.04904, 153.01854
F13 Hyperin a (A18) 30.35 C21H20O12 + 465.10275 465.10132 −1.432 465.10117, 303.04898, 153.12683, 135.11655, 85.02870
F14 Quercetin a (A19) 30.98 C15H10O7 + 303.04993 303.04932 −2.010 303.04901, 257.04413, 207.10074, 165.01768
F15 Hyperin-isomer 30.99 C21H20O12 + 465.10275 465.10165 −1.102 465.10165, 303.04926, 153.12700,135.11655, 85.02870
F16 Luteolin-glc-isomer 31.08 C21H20O11 + 449.10784 449.10660 −2.756 449.17566, 287.05414
F17 Luteolin-O-glc a (A20) 31.38 C21H20O11 + 449.10784 449.10645 −3.090 449.17886, 391.20599, 287.05432,
F18 Kaempferol-O-Glc-isomer 33.23 C21H20O11 + 449.10784 449.10660 −2.756 287.05408;153.01796, 145.04919, 127.03870,
F19 Kaempferol-rut 33.23 C27H30O15 + 595.16575 595.16406 −2.834 287.05405, 129.05443, 85.02871
F20 Kaempferol-O-Glc-isomer 34.02 C21H20O11 + 449.10784 449.10641 −3.179 287.05402, 153.01796, 121.02817
F21 Kaempferol-O-glu/gal + glu A 34.03 C28H32O16 + 625.17631 625.17426 −3.281 479.11679,317.06464
F22 Kaempferol-O-Glc-isomer 34.27 C21H20O11 + 449.10784 449.10687 −2.155 287.05420, 127.03868, 145.04906
F23 Luteolin/kaempferol-isomer 37.69 C15H10O6 + 287.05501 287.05417 −2.942 269.04379, 247.09439, 165.01772, 121.02880,
F24 Quercetin-isomer 41.79 C15H10O7 + 303.04993 303.04898 −3.132 303.04901,257.04413,153.01775, 165.01776, 105.03358
F25 Luteolin a (A25) 41.91 C15H10O6 + 287.05501 287.05408 −3.256 153.01747, 137.09558, 135.04381
F26 Naringenin a (A27) 45.76 C15H12O5 + 273.07575 273.07495 −2.930 273.07489, 153.01787, 147.04372
F27 Apigenin a (A29) 46.95 C15H10O5 + 271.06010 271.05927 −3.062 271.05923, 153.01784, 119.04916
F28 Kaempferol a (A31) 47.84 C15H10O6 + 287.05501 287.05411 −3.151 258.05060, 153.01787,133.02806, 121.02821
O1 Succinic acid 2.29 C4H6O4 − 117.01933 117.018179 −9.832 117.01817, 99.00740,73.02811
O2 Gallic acid a (A1) 2.97 C7H6O5 − 169.01425 169.01289 −8.026 169.01306, 125.02301
O3 5-Hydroxymethylfurfural a (A2) 2.98 C6H6O3 − 125.02442 125.02327 −9.198 125.02305, 97.02824,69.03322
O4 1¢-O-galloylsucrose 3.32 C19H26O15 − 493.11989 493.11902 −1.771 313.05569, 169.01317
O5 Matrine a (A3) 3.37 C15H24ON2 + 249.19614 249.19579 −1.404 249.19540, 232.15358
O6 6¢-O-galloylsucrose isomer 3.58 C19H26O15 − 493.11989 493.11880 −2.217 313.05627, 169.01279
O7 6¢-O-galloylsucrose isomer 3.78 C19H26O15 − 493.11989 493.11856 −2.704 313.05624, 169.01260
O8 Salvianic acid A sodium a (A4) 4.59 C9H9O5Na + 221.04214 221.04155 −0.495 221.07767, 205.15816, 175.14740, 111.08044
O9 Safflochalconeside isomer 4.67 C21H20O10 + 433.11292 433.11172 −2.778 415.10101, 235.02301
O10 Vanillic acid isomer 4.89 C8H8O4 − 167.03498 167.03358 −8.394 167.03377, 149.02299, 139.03867, 123.04369
O11 Safflochalconeside isomer 4.97 C21H20O10 + 433.11292 433.11194 −2.270 433.11240, 415.10089, 385.09070, 367.08029, 355.08008, 235.02301
O12 Tanshinol a (A5) 4.98 C9H10O5 − 197.04555 197.04436 −6.022 197.04468,179.03392,135.04376, 123.04372
O13 Ethyl gallate isomer 4.98 C9H10O5 − 197.04555 197.04436 −6.022 197.04468, 179.03392, 135.04376,123.04371
O14 Protocatechuic acid a (A6) 5.56 C7H6O4 + 155.03389 155.03360 −1.840 155.0348, 137.02309, 111.04404
O15 Ethyl gallate isomer 7.74 C10H12O5 − 211.06120 211.0007 −5.338 211.06024, 196.03683, 181.04944, 163.03877, 151.03871, 148.01527, 136.01520
O16 p-Anisicacid(4-MethoxybenzoicAcid)-isomer 7.97 C8H8O3 − 151.04007 151.03857 −8.590 151.03877, 133.02815, 123.04373, 107.04879
O17 Chlorogenic acid isomer 8.28 C16H18O9 − 353.08781 353.08694 −2.451 353.08737, 191.05504, 179.03381, 135.04372
O18 Methyl gallate 8.28 C8H8O5 − 183.02990 183.02856 −7.303 183.02872, 168.00516, 163.03880,135.04372
O19 Protocatechuic aldehyde isomer 9.54 C7H6O3 − 137.02442 137.02318 −9.049 137.02306, 93.03311
O20 Protocatechuic aldehyde isomer 9.54 C7H6O3 − 137.02442 137.02324 −8.612 137.02306, 109.02814,93.03306
O21 Protocatechuic aldehyde isomer 10.52 C7H6O3 − 137.02442 137.02323 −8.685 137.02301, 93.03305
O22 Tetramethylpyrazine a (A8) 11.29 C8H12N2 + 137.10733 137.10713 −1.422 137.10707, 122.08335
O23 Vanillic acid isomer 15.46 C8H8O4 − 167.03498 167.03360 −8.274 167.03378, 123.04375
O24 Benzoic Acid 15.86 C7H6O2 − 121.02950 121.02841 −9.006 121.02808, 108.02028,94.02835
O25 Chlorogenic acid a (A10) 17.12 C16H18O9 − 353.08781 353.08688 −2.621 353.08743, 191.05505, 179.03372, 135.04366
O26 Caffeic acid a (A11) 17.49 C9H8O4 − 179.03498 179.03365 −7.440 179.03389, 135.04378
O27 Safflor yellow A 18.50 C27H30O15 + 595.16575 595.16364 −3.539 577.15753,433.11160,147.04370
O28 Safflochalconeside isomer 18.50 C21H20O10 + 433.11292 433.11124 −3.886 415.10135, 235.02301
O29 Carthamidin/isocarthamidin-glu/gal isomer 18.50 C21H22O11 + 451.12349 451.12189 −3.542 289.06970, 271.05914, 211.02304
O30 Carthamidin/isocarthamidin-2glu/gal 18.50 C27H32O16 + 613.17631 613.17456 −2.856 451.12244,331.08035,289.06982,211.02307
O31 Hydroxysafflor yellow A a(A13) 18.76 C27H32O16 − 611.16176 611.15911 −2.418 611.16290,491.11926,473.10776,403.10294,325.07040, 295.06198
O32 Chlorogenic acid isomer 18.92 C16H18O9 − 353.08781 353.08670 −3.130 353.08759, 191.05508, 179.03381
O33 4-Hydroxytoluene; (4-Methylphenol) 19.70 C7H8O − 107.05024 107.04921 −9.622 107.04878
O34 Tanshinol isomer 23.84 C9H10O5 − 197.04555 197.04437 −5.972 197.04466, 169.01309, 125.02301
O35 Ethyl gallate isomer 23.84 C9H10O5 − 197.04555 197.04443 −5.667 197.04466, 169.01309,125.022297
O36 Safflochalconeside isomer 25.48 C21H20O10 + 433.11292 433.11157 −3.124 415.10184,397.09003,367.08011, 277.03345
O37 Coniferyl aldehyde, (ferulaldehyde) 26.03 C10H10O3 − 177.05572 177.05432 −7.949 177.05499, 162.03194, 149.05939, 129.01839, 105.03781, 99.00752,71.01251
O38 Ferulic Acid a (A16) 27.09 C10H10O4 + 195.06519 195.06482 −1.873 195.06458, 177.05418, 145.02809, 135.04388
O39 Vanillin 28.63 C8H8O3 − 151.04007 151.03857 −9.914 151.03877, 135.00745, 109.02804
O40 p-Anisic acid (4-Methoxybenzoic Acid) 28.63 C8H8O3 − 151.04007 151.03856 −9.980 151.03877, 135.00745, 109.02804
O41 Protocatechuic aldehyde a (A7) 28.68 C7H6O3 − 137.02442 137.02314 −9.341 137.02304, 93.03307
O42 Carthamidin/isocarthamidin-glu/gal isomer 28.76 C21H22O11 + 451.12349 451.12207 −3.143 289.06793, 169.01270, 147.04370
O43 Tetragalloyl glucose 28.81 C34H28O22 − 787.09994 787.09705 −3.679 787.09833,465.06750, 295.04462, 169.01303
O44 Carthamidin/isocarthamidin-glu/gal isomer 31.70 C21H22O11 + 451.12349 451.12238 −2.456 289.06793, 169.01265,147.04366, 85.02869
O45 Pentagalloylglucose 32.83 C41H32O26 − 939.11989 939.10724 −3.902 939.11371,769.08820, 617.08051, 447.05655, 295.04709, 169.01294
O46 Lithospermic acid isomer 33.03 C27H22O12 − 537.10385 537.10175 −3.908 537.10107,519.09387,375.06934,339.05014,295.06058,201.01610,179.03391,161.02318
O47 Lithospermic acid isomer 33.75 C27H22O12 − 537.10385 537.10022 −6.757 537.09991,375.06915,357.05890,201.01595, 179.03377
O48 Azelaic acid 33.95 C9H16O4 − 187.09758 187.09633 −6.693 187.09651, 169.08598, 143.10638, 125.09573
O49 Carthamidin/isocarthamidin-glu/gal isomer 35.33 C21H22O11 + 451.12349 451.12234 −2.545 304.09924, 289.06793, 169.01265
O50 Lithospermic acid isomer 35.77 C27H22O12 − 537.10385 537.10040 −6.422 375.06982, 201.01608, 179.03377, 135.04445
O51 Rosmarinic acid a (A22) 35.88 C18H16O8 − 359.07724 359.07632 −2.564 359.07629, 197.04462, 179.03386, 161.02316
O52 Salvianolic acid A isomer 37.03 C26H22O10 − 493.11347 493.11267 −2.738 295.06073,197.04436,179.03427,109.02807
O53 Lithospermic acid isomer 37.03 C27H22O12 − 537.10385 537.10297 −1.637 295.06070, 179.03328, 109.02803
O54 Crocin I a (A23) 38.77 C44H64O24 − 975.37148 975.37061 −1.849 651.26538, 327.16083, 283.17017
O55 Salvianolic acid B a (A24) 39.18 C36H30O16 − 717.14611 717.14417 −2.702 519.09308,339.05045,321.04007,295.06061, 249.05511
O56 3,7- or 3,8-Dimethyl ellagic acid isomer 42.65 C16H10O8 − 329.03029 329.02939 −2.737 329.03018, 314.00659,298.98291
O57 Salvianolic acid A a (A26) 43.42 C26H22O10 − 493.11347 493.11261 −2.859 295.06076,185.02339, 109.02803
O58 3,7- or 3,8-Dimethyl ellagic acid isomer 43.57 C16H10O8 − 329.03029 329.02945 −2.554 329.02982, 314.00644, 298.98282, 270.98758
O59 Ethyl4-hydroxy-3-methoxycinnamate 44.31 C12H14O4 − 221.08193 221.08087 −4.805 221.08109, 177.09096
O60 Salvianolic acid C isomer 44.92 C26H20O10 − 491.09837 491.09723 −2.321 311.05563, 293.04517
O61 Paeonola(A28) 46.25 C9H10O3 + 167.07027 167.07008 −1.142 167.06992,149.05942,121.06463,109.02848
O62 Salvianolic acid C isomer 48.31 C26H20O10 − 491.09837 491.09741 −1.955 311.05563, 293.04517
O63 Ethyl ferulatea(A32) 50.72 C12H14O4 − 221.08193 221.08078 −5.212 221.08099,177.09093,149.09587,134.03592121.02803, 71.04876,69.03313
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a:Structures confirmed by comparison with reference standards, and A1-A38 were the mark number of reference standards.

Bold characters: the base peaks in MSn spectra.

Identification of monoterpene glycosides in XBJ

19 monoterpene glycosides were identified and listed in Table 1. M1 gave [M−H]− ion at m/z 359.13400 (C16H23O9) in full scan mass spectrum. In it’s MS/MS2 experiment, the obtained ion produced characteristic fragment of [M−H−Glc]− at m/z 197.08099 (C10H13O4), corresponding to the paeonisuffrone, was observed, the further loss of H2O group generated the fragment of [M−H−Glc−H2O]− at m/z 179.07028 (C10H11O3) was also observed. Thus, M1 was deduced as 1-O-β- d-glucopyranosyl-paeoni-suffrone. Three isomers (M4, M5 and M7) revealed the same [M−H]− ions at m/z 527.13922 (C23H27O14). In the MS/MS2 experiment of M4, the [M−CH2OH]− ion at m/z 497.12943 (C22H25O13) and [M−CH2OH−H2O]− ion at m/z 479.11896 (C22H23O12) was produced by the loss of CH2OH unit, and the further loss of H2O. The precursor ion of M4 generated fragment at m/z 313.05627 (C13H13O9) by loss of the aglycone moiety, and the further loss of hexose moiety produced the galloyl fragment at m/z 169.01294 (C7H5O5). M5 had the same ions at m/z 313.05627 and 169.01294 in its MS/MS spectrum with M4 and M7. M4, M5 and M7 were identified as 6′-O-galloyl Desbenzoylpaeoniflorin and its isomers.

Four isomers (M12, M14, M15 and M16) revealed the same [M−H]− ions at m/z 631.16498 (C30H31O15). In the MS/MS spectrum of M12, the loss of H2O group from precursor ion at m/z 613.15570 (C30H29O14) and the further loss of benzoyl group at m/z 491.11874 (C23H23O12) was observed. The obtained ion produced fragment corresponding to galloyl attached at one hexose moiety at m/z 313.05603 (C13H13O9), and the galloyl fragment at m/z 169.01294 (C7H5O5) were found. All of M14, M15, and M16 had the same fragments at m/z 313.05603 (C13H13O9), at m/z 169.01294 (C7H5O5), and benzoyl group at m/z 121.02809 (C7H5O2) in their respective MS/MS spectra. M12, M14, M15, and M16 were identified as galloylpaeoniflorin and its isomers. M17 and M18 showed the same [M−H]− ion at m/z 599.17554 (C30H31O13). Apart from the characteristic fragments of paeoniflorin, both of their MS/MS spectra displayed the fragment at m/z 477.13870 (C23H25O11), 281.06613 (C13H13O7), 137.02303 (C7H5O3), 121.02802 (C7H5O2), indicating the existence of O-benzoyl unit, benzoyl unit, and hexose moiety. M17 and M18 were deduced as benzoyloxypaeoniflorin and its isomer. M19 displayed the [M−H]− ion at m/z 583.18210 (C30H31O12), which had one less oxygen than that of M17 and M18. By comparing and the analysis of their MS/MS spectra, the absence of ion at m/z 137.02303 (C7H5O3), and the presence of ion at m/z 121.02801 (C7H5O2), indicated the benzoyl unit in M19 instead of O-benzoyl unit in M17 and M18. Thus, M19 was identified as benzoylpaeoniflorin.

Identification of phenanthrenequinone in XBJ

19 phenanthrenequinone were identified and listed in Table 1. P1 displayed a [M + H]+ ion at m/z 297.11133 (C18H17O4). In the MS/MS2 experiment, the obtained ion produced [M−H2O]+ fragment at m/z 279.15570(C18H15O3) and [M-2H2O]+ ion at m/z 261.09055 (C18H13O). P1 was identified as tanshinone VI. Four isomers P3, P4, P5, and P14 revealed the same [M + H]+ ion at m/z 313.14249 (C19H21O4). In the MS/MS2 experiment, the obtained ion produced [M−H2O]+ fragment at m/z 295.13196 (C19H19O3), [M−CO2]+ ion at m/z 269.15262 (C18H21O2), and [M − H2O−CO2]+ ion at m/z 251.14232 (C18H19O) were found in P3. P14 had the same fragment ions with P3. In the MS/MS spectrum of P4, [M−H2O]+, [M−2H2O]+, and [M−H2O−CO2]+ ions at m/z 295.13177 (C19H19O3), m/z 277.12137 (C19H17O2), m/z 251.14224 (C18H19O3) were detected. The fragment of [M−CO]+, and [M−CO−H2O]+ ions at m/z 285.14700 (C18H21O3), and m/z 267.13751 (C18H19O2) were observed in the MS/MS2 experiment of P5. This fragmentation information were similar with that of phenanthrenequinone, and their molecular was accordance with tanshinone II B, the major constituent in tanshin, one composition of traditional Chinese medicine in XBJ. Thus, P3, P4, P5, and P14 were deduced as tanshinone II B and its isomers.

P8 gave a [M + H]+ ion at m/z 299.16432 (C19H23O3). Its MS/MS experiment generated [M−H2O]+ ion at m/z 281.15341 (C19H21O2), and the further loss of CO produced the [M−H2O−CO]+ ion at m/z 253.15788 (C18H21O). P8 was identified as miltiodiol. P9 exhibited a [M + H]+ ion at m/z 299.16339 (C19H23O3) in the positive full scan mode. The fragment at m/z 281.15314 (C19H21O2) indicated the loss of H2O from the precursor ion. The other product ion at m/z 253.15788 (C18H21O) revealed the further splitting of a CO2 group. This information led to the conclusion that P9 was deoxyneocryptotanshinone. Two isomers P13 and P18 showed the same [M + H]+ ion at m/z 297.14852. The MS/MS experiment of P13 generated [M−CO2]+ ion at m/z 249.09039 (C18H21O). P13 were identified as Cryptotanshinone by comparing with the retention time and high-resolution accurate mass and P18 was identified as its isomers.

Identification of lactones in XBJ

The detailed MS data of 33 lactones were listed in Table 1. Four isomers L1, L4, L5 and L9 revealed the same [M + H]+ ions at m/z 227.12724 (C12H19O4). In their MS/MS spectrum, the characteristic fragment ions of senkyunolide J/N, such as 209.11671 (C12H17O3), 191.10614 (C12H15O2,), 163.11134 (C11H15O), 153.05424(C8H9O) were observed. So they were assigned as senkyunolide J/N and its isomers. Similarly, L2, L3, L8 and L10 were identified as senkyunolide I/H and its isomers owing to the presence of diagnostic fragment ions related to senkyunolide I/H. L7, L11, and L12 showed the same [M + H]+ ion at m/z 207.10114 (C19H21O4) in their full scan positive mass spectrum. In their MS/MS spectra, the same ions at m/z 189.09053 (C12H13O2) produced by the loss of H2O group from precursor ion, and m/z 161.09546 (C11H13O) produced by the further loss of CO group were found. These characteristic information related to senkyunolide suggested L7, L11, and L12 to be senkyunolide F and its isomers. L13, L15, and L23 were determined as senkyunolide B/C/E, for the characteristic fragment ions of senkyunolide B/C/E, at m/z 187.07489 (C12H11O2), 177.09053 (C11H13O2,), and 163.03853 (C9H7O3), 149.02296 (C8H5O3). L18 exhibited [M + H]+ ion at m/z 209.11652 (C12H17O3) in its full scan positive mass spectrum, indicating the molecular formula of C12H16O3. The MS/MS experiment of L18 generated [M−2H2O]+ ion at m/z 173.09526 (C12H13O) by successive loss of H2O group from the precursor ion. L18 was identified as senkyunolide G/K. L21, L22, L26 and L28 exhibited the same [M + H]+ ion at m/z 193.12196 (C12H17O2) in positive ion mode, indicating the molecular formula of C12H17O2, which had one less oxygen atom than that of L18. They had one less hydroxyl than that of L18 in the structure, which was verified by the fragments at m/z 175.11171 (C12H15O), m/z 165.12669 (C11H17O), m/z 137.05954 (C8H9O2) in their MS/MS spectra. Thus, L21, L22, L26, and L28 were identified as senkyunolide A and its isomers. The same [M + H]+ ions at m/z 279.15839 (C16H23O4) of L24, L25, and L27 revealed the molecular formula of C16H22O4. The fragment ions at m/z 261.14850 (C16H21O3), 233.15289 (C15H21O2), 215.14252 (C15H19O), were generated by loss of H2O, further loss of CO, and further loss of H2O, respectively. The ion at m/z 191.10616 (C12H15O2) was produced by three times of successive loss of H2O from fragment of [M-H2O-CO]+ at m/z 233.15289 (C15H21O2), and the further loss of H2O generated ion at m/z 173.09566 (C12H13O). These ions are the characteristic neutral losses associated with the senkyunolide M. Thus, L24, L25 and L27 were indicated as senkyunolide M and its isomers.

Identification of flavonoids in XBJ

28 flavonoids were detected and deduced in positive ion mode. The detailed fragmentation information of flavonoids was listed in Table 1. F7, F12 and F9 displayed the same [M + H]+ ion at m/z 611.15912 (C27H31O16), and they were deduced as rutin and its isomers, based on the presence of diagnostic fragment ions at m/z 303.04904 (C15H11O7), 153.01854 (C7H5O4). Five isomers of F8, F16, F18, F20 and F22 displayed the same [M + H]+ ion at m/z 449.10632 (C21H21O11) with luteolin-O-glc of F17. In their MS/MS spectra, by lossing of the hexose moiety generated the [M−H−Glc]+ ion at m/z 287.05411 (C15H11O6), corresponding to the aglycone of kaempferol or luteolin. F8, F16, F18, F20, and F22 were identified as hexose glycoside of kaempferol or its isomers. F23, F25 and F28 showed the same [M + H]+ ion at m/z 287.05420 (C15H11O6). In their MS/MS spectra, the characteristic fragments at m/z 153.01807 (C7H5O4), 133.02815 (C8H5O2), and 121.02580 (C7H5O2), which were produced by the two different reaction routines of RDA cleavage, were observed. The loss of CO moiety from precursor ion generated the ion m/z 258.05179 (C14H10O5) was also found. Comparing with the retention time of reference solution, F25 was confirmed as luteolin and F28 was kaempferol. The characteristic ions of kaempferol are m/z 258.05060, 153.01787, 133.02806 and 121.02821, and the characteristic ions of luteolin are m/z 153.01747, 137.09558 and 135.04381.

Identification of phenolic acids and other compounds in XBJ

The MS data of 63 detected phenolic acid and other compounds were listed in Table 1. O12 and O34 showed the same [M−H]− ion at m/z 197.04436 (C9H9O5) in the negative full scan mode. Both of their MS/MS spectra displayed the [M−H2O]− and [M−H2O−COOH]− ions at m/z 179.03392 (C9H7O4) and 135.04376 (C8H7O2) suggested that O12 and O34 were Tanshinol and its isomer. O17, O25 and O32 displayed the same [M−H]− ion at m/z 353.08688 (calculated 353.08781, error, −2.621 ppm) in negative full scan mode. Their MS/MS spectra showed similar ions at m/z 179.03372 (C9H7O4) and 135.04366 (C8H7O2). This fragmentation was associated with that of caffeic acid. O17, O25 and O32 were identified as chlorogenic acid and its isomers. Four isomers of O19, O20, O21 and O41 showed the same [M + H]+ ion at m/z 137.02442(C7H6O3). The MS/MS experiment of O41 generated [M−CO2]+ ion at m/z 93.03307 (C6H5O). O41 were identified as Cryptotanshinone by comparing with reference, and the O19, O20 and O21 were identified as its isomers. In the negative full scan mode, O26 showed [M−H]− ion at m/z 179.03365 (C9H7O4). The MS/MS experiment yielded [M−COOH]− ion at m/z 135.04378 (C8H7O2). O26 was identified as caffeic acid. O53 showed [M−H]− ion at m/z 359.07632 (C18H15O8). In the MS/MS experiment, the ion at m/z 179.03386 (C9H7O4) was triggered by the loss of caffeic acid residue. Further fragment at m/z 197.04462 (C9H9O5) suggested the existence of acid. Therefore, O53 was identified as rosmarinic acid.

Quantitative analysis of samples

A thorough and complete method validation for assaying 38 bioactive compounds in XBJ was done referring to ICH guidelines24. The UHPLC-Q-Orbitrap mass spectrometry was validated with respect to linearity, sensitivity, accuracy and precision, reproducibility and stability.

Method Validation

Linearity, LOD and LOQ

Standard stock solutions containing 38 analytes were prepared and diluted to seven appropriate concentrations for the construction of the calibration curves. Each solution was injected in triplicate, and then the linear regression equation was obtained by plotting the analyte peak area (Y) vs a series of analyte concentrations (X). The regression equation, coefficient of determination (R 2) and linear range are given in Supplementary Table S1. All the analytes showed good linearity with R 2 more than 0.9994 in the concentration range. The LOD and LOQ under the optimized chromatographic conditions were evaluated at a signal-to-noise ratio (S/N) of 3 and 10, respectively. The values of LODs and LOQs were in the range of 0.01~35.77 ng·mL−1 and 0.03~119.22 ng·mL−1, respectively (Supplementary Table S1).

Accuracy and precision

The precision of the established method was evaluated by intra-day and inter-day variability, and the relative standard deviations (RSD) were taken as a measure. The mixed standard solution at middle concentrations was analyzed in six replicates within one day and on 3 consecutive days. The results are shown in Supplementary Table S1, and the RSD values of the intra-day and inter-day of 38 compounds were all less than 2.97%, which showed good precision of the developed method.

The accuracy of the established method was evaluated by recovery test and RE (relative error). The samples were spiked with three concentration levels (80, 100, and 120%) of known amounts of 38 reference compounds. The spiked samples of each concentration were analyzed in triplicate. The accuracy was calculated as the quotient of the measurement and the nominal value of the analyte added to the sample. The detailed accuracy data is presented in Supplementary Table S2. The mean recoveries were ranged from 98.5% to 101.3% with RSDs less than 2.98%.

Reproducibility and Stability

In order to confirm the reproducibility, six different samples from the same batch sample were analyzed within one day and on three consecutive days. The RSDs were used as a measure and the acceptance criterion should be within 5.0%. The results are shown in Supplementary Table S1 and the RSD values of 38 compounds were all less than 3.0%, which showed good reproducibility of the developed method.

The stability of the sample solution was analyzed at room temperature on three consecutive days. The stability of the standard solutions stored at 4 °C was also examined on three consecutive days. Injections were performed at 0, 12 hour, 1, 2, 3, 5, and 7 days. The stability RSD values of 38 compounds in the sample solution were all less than 2.86% and those in standard solutions were all less than 2.0%, which showed that all analytes in the sample solution (at room temperature) and the standard solutions (at 4 °C) were found to be very stable.

Analysis of chemical profile of XBJ sample

The developed UHPLC-Q-Orbitrap HRMS method was adopted for the routine screening of the 38 bioactive compounds in 10 XBJ samples. 38 bioactive compounds were unambiguously identified by comparing the retention times and high-resolution accurate mass of reference standards. The polarity switching in full scan modes of UHPLC-Q-Orbitrap HRMS was used to achieve the highest response intensities of various types of constituents. In addition, the Q-Orbitrap HRMS as a powerful high resolution mass spectrometry, has the function of qualitative and quantitative simultaneously, namely compounds could be qualitative and quantitative in one analysis. Table 2 showed the obtained quantitative results of each compound calculated according to calibration curves. The results shows that two compounds (Hydroxysafflor yellow A and Paeoniflorin) are the predominant constituents obviously, the contents of which are much higher than other compounds. Hydroxysafflor yellow A and Paeoniflorin are two major marker components in Carthami Flos and Paeoniae Radix Rubra. Moreover, the Q-Orbitrap HRMS has very high sensitivity, so the low-content compounds, such as Levistolide A, Tetramethylpyrazine, Butylidenephthalide and Tanshinone I, were investigated simultaneously. Thus, the constituents with high and low levels contents could be quantified in one analysis.

Table 2.

Quantitative analytical results for 38 compounds in XBJ from 10 batches (n = 3, μg/mL).

Compounds 1500181 1504101 1504111 1504121 1505211 1505671 1508171 1508191 1509082 1509132
A1 7.203 6.266 5.557 5.343 6.995 5.435 5.179 6.339 7.117 6.886
A2 14.537 12.644 10.790 10.827 13.447 14.746 10.530 12.477 11.308 13.658
A3 0.086 0.099 0.145 0.099 0.116 0.095 0.095 0.150 0.105 0.118
A4 0.806 0.742 0.629 0.636 0.697 0.706 0.731 0.699 0.823 0.731
A5 3.010 2.840 2.592 2.396 3.122 3.060 2.789 2.910 2.695 2.892
A6 4.471 4.354 4.214 4.355 4.196 4.023 4.130 3.929 3.632 4.536
A7 4.974 4.430 4.480 4.138 4.164 4.890 4.654 4.202 4.521 4.733
A8 0.004 0.003 0.003 0.004 0.003 0.003 0.004 0.005 0.004 0.004
A9 4.334 4.508 4.739 5.567 4.895 5.181 6.423 4.640 6.459 6.253
A10 3.398 3.618 3.521 3.107 2.778 3.202 3.904 3.601 3.701 3.254
A11 4.733 5.033 5.057 4.366 4.619 4.923 5.071 5.170 4.467 4.848
A12 36.136 38.274 39.614 37.579 38.264 38.977 37.833 37.461 38.625 38.148
A13 587.385 599.696 608.705 550.898 517.190 614.478 608.009 620.303 590.146 593.456
A14 16.582 19.526 17.490 15.570 20.358 15.286 17.695 17.465 20.055 15.971
A15 893.515 925.366 880.513 870.379 874.547 906.911 886.107 884.818 818.990 875.418
A16 30.993 29.554 28.278 25.810 29.195 26.904 29.529 30.674 31.704 29.231
A17 4.010 3.723 4.159 3.394 3.326 3.765 3.470 3.267 3.413 3.732
A18 0.404 0.420 0.503 0.419 0.497 0.487 0.458 0.404 0.446 0.366
A19 1.224 1.230 1.297 1.003 1.057 1.075 0.998 1.093 0.871 1.117
A20 1.115 1.144 1.271 1.020 0.930 0.995 0.932 0.904 1.176 1.037
A21 85.282 84.158 84.915 84.707 85.356 93.482 90.544 79.375 83.856 83.856
A22 5.960 5.392 5.522 4.644 5.406 5.376 6.157 6.139 6.115 5.783
A23 1.654 1.882 1.800 1.517 1.520 1.546 1.518 1.620 1.669 1.669
A24 2.261 2.090 1.940 1.595 2.017 2.164 2.344 2.448 2.377 2.067
A25 0.124 0.105 0.126 0.123 0.106 0.119 0.109 0.131 0.124 0.117
A26 0.039 0.047 0.047 0.037 0.049 0.041 0.059 0.039 0.037 0.040
A27 0.350 0.495 0.476 0.418 0.360 0.328 0.412 0.376 0.386 0.330
A28 0.025 0.029 0.020 0.025 0.022 0.025 0.024 0.023 0.020 0.027
A29 0.451 0.458 0.459 0.393 0.424 0.433 0.421 0.433 0.403 0.403
A30 30.026 35.814 33.435 30.979 35.207 31.965 30.470 29.264 29.210 31.023
A31 0.206 0.213 0.219 0.231 0.242 0.221 0.200 0.236 0.282 0.205
A32 0.369 0.368 0.398 0.383 0.387 0.379 0.381 0.382 0.477 0.375
A33 0.196 0.246 0.250 0.194 0.183 0.209 0.176 0.187 0.179 0.148
A34 0.019 0.015 0.017 0.017 0.020 0.016 0.017 0.020 0.015 0.014
A35 0.020 0.021 0.021 0.016 0.022 0.020 0.022 0.018 0.023 0.023
A36 0.473 0.561 0.566 0.515 0.586 0.525 0.588 0.543 0.555 0.524
A37 0.00046 0.00047 0.00049 0.00047 0.00058 0.00055 0.00057 0.00046 0.00054 0.00050
A38 0.084 0.078 0.080 0.080 0.078 0.088 0.069 0.085 0.072 0.085
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The RSD of total amounts of investigated 38 compounds in 10 batches XBJ samples was 2.81%, which showed good stability of the total content. However, significant variations were observed as well. The RSD of each compound in 10 batches XBJ samples was in range of 2.48% to 19.43%, which showed instability of the some compounds. However, multiple active components, including macro- and micro-components, are frequently considered to be responsible for the therapeutic effects25. So, the present analysis of multiple components is more reasonable for quality control of XBJ injection.

Quality assessment of XBJ with the established strategy

Fingerprinting

Fingerprinting strategies are internationally accepted as an acceptable means of quality control (QC) for TCMs26. There are significant advantages of using fingerprinting strategies for sample differentiation, as fingerprinting not only determines the characteristic patterns of each plant type but also reveals the inherent relationships between multiple compounds. The good precision, reproducibility, stability of UHPLC-Q-Orbitrap HRMS analysis were demonstrated. The chromatograms Xcalibur raw files of ten batches sample was imported into the SIEVE software. The batch of 1500181 was selected as reference chromatogram. In order to focus on the most effective information, time windows of 0–60 min was selected to generate chromatographic fingerprinting. The similarity values obtained by SIEVE software was calculated through the overall evaluation of 10 batches total ion current chromatograms. The identical peaks in 10 batches sample chromatograms can be matched in automatic and proceeded peak alignment. The retention time and peak area of all peak in 10 batches sample make a comparison with the reference chromatogram. The correlation coefficients of all introduced chromatograms relative to that of reference chromatogram would be calculated. The similarity values of 10 samples (No.1500181, 1504101, 1504111, 1504121, 1505211, 1505671, 1508171, 1508191, 1509082 and 1509132) in fingerprintings in positive and negative mode were 1, 0.990, 0.988, 0.990, 0.991, 0.989, 0.991, 0.988, 0.977 and 0.993, respectively. The similarity values were all more than 0.9 in positive and negative mode, which indicated that the samples from different batches had strong similarities with high correlation coefficients of similarities. To some degree, this results demonstrate that the fingerprinting chromatograms of these samples might be used to assess the quality of XBJ injection.

Principal component analysis

PCA was used to further classify the 10 samples. PCA is an analytical method that is used to reduce a large set of variable into a smaller set of “artificial”variables known as principal components (PCs), which account f or most of the variance in the original variables. In the present analysis, the data matrix of ten batches samples and 38 bioactive compounds was imported into the multivariate statistical analysis software SIMCA 14.0. The PCA-X model was adopted to match the data and the original 38 variable dimension generated 2 new variables through software automatically, that is the two principal components. After the data fitting, the principal component 1 of variable was accounted for larger percentage of 63.8%, which could reflect the main characteristics of the original data. So PC1 would be suitable for revealing correlations among the different variables. The Score Scatter Plot (Fig. 4A) is used to evaluate the stability of 10 batches XBJ samples. The deviation represented the degree of stability. The deviation represented the degree of stability. The smaller the deviation in the PC1 axis, the better stability. The Fig. 4A shows the bias of 10 batches was within ± 2 SD, indicating the quality of 10 batches was more stable. In addition, the bias of 8 batches in 10 batches was within ± 1 SD, while the bias of 2 batches in 10 batches was ranged from ± 1 SD to ± 2 SD.

Figure 4.

Figure 4

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(A) PCA Score Scatter Plot. (B) PCA Loading Column Plot.

The PCA Loading Plot could reflect the weight size of original variable in the principal component analysis. The greater the absolute value of original variable in the PCA Loading Plot, the more importance role of original variable in the overall distribution. So the PCA Loading Plot can make it possible to discover the variables leading to the difference. In the Loading Column Plot (Fig. 4B) of the scores, the variables of 13 and 15 were the farthest from the origin on the PC1 and PC2. A13 and A15 as the important quality markers, has a relationship with different batch of drugs on the scatter plot distribution location. In the Fig. 4B, A13 and A15 were positive and the absolute value is larger in Loading Plot on PC1, which make the most batch in the positive quadrant portion of the Score Scatter Plot and keep positively correlated with them. Because the level of A13 and A15 is lower than the average level, the batch 1505211 has obvious anomaly in the overall distribution. So the two components made this batch negatively correlated and this batch was spotted in the negative quadrant part of the Score Scatter Plot. That is to say that two markers responsible for the cluster formation were mainly compounds (A13 and A15) that suggested that the contents of Hydroxysafflor yellow A and Paeoniflorin had a significant relationship with quality of XBJ injection. In addition, the variables of 14, 21 and 30 had a certain statistical significance compared with other variables. The compounds were Albiflorin, Senkyunolide I/H and Benzoylpaeoniflorin, respectively. This three compounds could provide some reference meaning for quality evaluation of XBJ injection. In 2013 edition Drug Standards of China, Hydroxysafflor yellow A and Paeoniflorin were selected as markers due to the two highest levels chemical composition. However, the therapeutic effects are frequently considered to be connected with multiple active components, including macro- and micro-components. So, the five markers, Hydroxysafflor yellow A, Paeoniflorin, Albiflorin, Senkyunolide I/H and Benzoylpaeoniflorin, were more meaningful for the quality of XBJ injection.

Assay of the five markers in XBJ sample

An UPLC-MS/MS method was developed for the routine determination of five markers in XBJ samples within 5 minutes. And the method was validated according to the above section “Method validation”. Satisfactory linearity and correlation coefficient were achieved with linear ranges. The relative standard deviations of precisions, repeatability, stability and recovery were all meeting requirements. The UPLC-MS/MS method could apply for the analysis of five marks in XBJ samples. The typical chromatograms of a standard mixture of five markers (A) and an XBJ sample (B) are shown in Fig. 5. This UPLC-MS/MS method was simpler in operation and higher in data handling efficiency for widely application.

Figure 5.

Figure 5

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The typical chromatograms of a standard mixture of five markers (A) and an XBJ sample (B).

Methods

Reagents and materials

HPLC grade methanol and acetonitrile for qualitative analysis were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid of HPLC grade purchased from Aladdin Industrial Co., Ltd. (Shanghai, China). Ammonium acetate was MS grade and purchased from Anpel scientific instrument Corporation Ltd. (Shanghai, China). All other chemicals were of analytical reagent grade. Ultra-pure water (18.2 MΩ) was purified by Millipore system (Millipore, Shanghai, China) and all solutions were filtrated 0.22 μm pore size filters.

The reference standards of compounds A1-A38 were purchased from Chengdu Must Bio-technology Co., Ltd. (Sichuan, China). The purities of all the reference standards were over 98% and their chemical structures were illustrated in Fig. 2. Ten batches commercial patent medicines of XBJ were prepared by Tianjin Chase Sun Pharmaceutical Co., Ltd. (Tianjin, China).

Standard solution and samples preparations

The stock standard solutions of 38 reference standards were dissolved in methanol with concentration of 1.0 mg/mL for each compound, respectively. Then, each stock solution was mixed with 50% methanol to prepare a final mixed standard solution. A series of working standard solutions were prepared by the successive dilution of the mixture of standard solutions with 50% methanol. All the solutions were stored at 4  °C before use. Ten batches of commercial preparations of XBJ were directly subjected to UHPLC-MS analysis after being filtered through a 0.22 μm syringe filter.

Chromatographic conditions and Mass spectrometric conditions

In the quantitative analysis of 38 compounds, an UHPLC Dionex Ultimate 3000 with Q-Exactive hybrid quadrupole-orbitrap mass spectrometer system was utilized. Chromatographic peaks were separated on a Waters ACQUITY UPLC® HSS C18 column (2.1 mm × 100 mm, 1.8 μm) at a flow rate of 0.2 mL/min with gradient acetonitrile (A) and water containing 10 mM ammonium acetate (B) as follows: 0–10 min, 5% A, 10–45 min, 5–30% A, 45–60 min, 30–100% A, and then the column was re-equilibrated at 5% A for 2 min prior to the next injection. The injection volume was 5 μL for analysis. The Q-Exactive mass spectrometer was equipped with heat electrospray ionization (HESI), an online vacuum degasser, a quaternary pumps, an autosampler, a thermostated column compartment and ultraviolet detector (UV). The optimized parameters of mass spectrometry were illustrated as below: spray voltage: + 3.5 kV or −2.8 kV; sheath gas pressure: 40 arb; Aux gas pressure: 10 arb; sweep gas pressure: 0 arb; capillary temperature: 320 °C; auxiliary gas heater temperature: 300 °C; S-lens RF level: 50 V; scan mode: (1) full MS: Resolution: 70,000; automatic gain control (AGC) target: 3.0e;6 maximum injection time (IT): 200 ms; scan range: 80–1200 m/z; (2) dd-MS2/dd-SIM: Rsolution: 17,500; AGC target: 1.0 e5; maximum IT: 50 ms; Loop count: 5; Isolation window: 2.0 m/z; NCE/stepped: 20, 30, 40; Dynamic exclusion: 10.0 s. Nitrogen was used for spray stabilization and as the collision gas in the C-trap. All data collected in profile mode were acquired and processed using Thermo Xcalibur 3.0 software.

In the quantitative analysis of five marks, a Waters Xevo TQD UPLC-MS/MS system (Waters Corp., Milford, MA, USA) was employed. Chromatographic peaks were separated on a Waters ACQUITY UPLC® HSS C18 column (2.1 mm × 100 mm, 1.8 μm) at a flow rate of 0.2 mL/min with gradient acetonitrile (A) and water containing 10 mM ammonium acetate (B) as follows: 0–0.5 min, 5% A; 0.5–1.0 min, 5–20% A; 1–3.0 min, 20–30% A; 3.0–4.5 min, 30–100% A, 4.5–5.0 min, 100% A. A subsequent re-equilibration time (2 min) should be performed before next injection. The injection volume was 5 μL for analysis. The Waters Xevo TQD mass spectrometer with electrospray ion source (ESI) was used. The MS spectra were acquired in MRM mode using polarity switching. The capillary voltage was set to 3.5 kV, and the source temperature was maintained at 350 °C, nitrogen gas was used as desolvation gas 650 L/h and cone gas 50 L/h and argon gas was employed as collision gas. The most appropriate precursor-to-product ion pair, cone voltage (CV) and collision energy(CE) are listed in Table S3. All data was acquired and integrated by Masslynx V4.1 software.

Mass spectrometric conditions

Statistical data analysis

The fingerprinting was performed on different XBJ samples by SIEVE 2.0 software (Thermo Scientific, San Jose, USA), which was used for evaluating the similarities between different samples. The similarity was evaluated with the correlation coefficients, and the calculation of correlation coefficients was mainly based on the peak area and retention time. The base peak intensity chromatographic data obtained from the positive or negative ion UHPLC-Q-Orbitrap HRMS analyses were imported in the form of Xcalibur raw files into the SIEVE software. With SIEVE software, the chromatogram can be normalized, and the identical peaks in each chromatogram can be matched in automatic or manual mode. All the batches of XBJ samples were used to construct fingerprinting. Subsequently, the correlation coefficients of all introduced chromatograms relative to that of reference chromatogram would be calculated. In a word, the software made the analysis method accurate and rapid.

Principal component analysis (PCA) involves a mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components. This transformation is defined in such a way that the first principal component has as high a variance as possible or accounts for as much of the variability in the data as possible27. PCA is an unsupervised pattern recognition technique, which is a data visualization method useful for a rapid means of visualizing similarities or differences within multivariate data28. PCA makes it possible to represent objects or variables on a graph, with different objectives to study the proximity of objects in order to differentiate them and to detect atypical objects, and also to analyze the position of objects in varied representations. Thus, we could probably speculate the chemical components causing quality differences in different batches. The PCA was performed on different XBJ samples by SIMCA 14.0 software (Umetrics, Sweden).

Electronic supplementary material

Supplementary information (144.5KB, doc)

Acknowledgements

This work was supported by the Colleges and universities in Henan province key scientific research project (No. 18A360022), the Foundation of Beijing medical and health (No. YWJKJJHKYII-B16240) and the National Natural Science Foundation of China (No. 81370364). This work was also supported by the Foundation of the First Affiliated Hospital of Zhengzhou University.

Author Contributions

X.-J.Z. designed the research; T.-W.S. conducted the chemical preparation; Z.-S., L.-H.Z., J.-F.T. and L.Z. participated in the study design, experiments, data processing and preparation of the manuscript; L.-H.Z., Z.-S. and X.-J.Z. edited the manuscript; D.-L.D. and J.K. were involved in revision of the manuscript. All authors read and approved the final version of manuscript.

Competing Interests

The authors declare that they have no competing interests.

Footnotes

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41598-017-17170-y.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Lihua Zuo, Email: zuolihua2013@126.com.

Xiaojian Zhang, Email: zhangxiaojian_yxb@163.com.

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