Chemical Profiling of Kaliziri Injection and Quantification of Six Caffeoyl Quinic Acids in Beagle Plasma by LC-MS/MS

Vitiligo is a stubborn multifactorial skin disease with a prevalence of approximately 1% in the global population. Kaliziri, the seeds of Vernonia anthelmintica (L.) Willd., is a well-known traditional Uyghur medicine for the treatment of vitiligo. Kaliziri injections is a Chinese-marketed treatment approved by the China Food and Drug Administration for the treatment of vitiligo. The significant effects of Kaliziri injection have been thoroughly studied. However, chemical components studies and plasma quantification studies are lacking for Kaliziri injection. Ultra-high-performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry was employed to comprehensively characterize the caffeoyl quinic acid derivatives present in Kaliziri injection. Based on accurate mass measurements, key fragmental ions and comparisons with reference standards, 60 caffeoyl quinic acid derivatives were identified in Kaliziri injections, including caffeoyl quinic acids, coumaroyl caffeoyl quinic acids, dicaffeoyl quinic acids, feruloyl caffeoyl quinic acids, and dicaffeoyl quinic acid hexosides. Moreover, an HPLC-MS/MS method was developed and validated for the quantitative analysis of 5-caffeoyl quinic acid, 4-caffeoyl quinic acid, 1,3-dicaffeoyl quinic acid, 3,4-dicaffeoyl quinic acid, 3,5-dicaffeoyl quinic acid and 4,5-dicaffeoyl quinic acid in beagle plasma. The quantitative HPLC-MS/MS method was applied to quantify these six major caffeoyl quinic acids in beagle plasma after the subcutaneous administration of Kaliziri injection. All of the six analytes reached their peak plasma of concentrations within 30 min.


Introduction
Vitiligo, caused by the loss of the function of melanocytes and melanin in skin and hair, is an autoimmune disease characterized by the appearance of white spots, with an estimated prevalence of approximately 1% in the global population [1,2]. Clinical and epidemiological investigations have shown that vitiligo is a complex multifactorial disease [3].
Contemporary treatment strategies for vitiligo include phototherapy, local or systemic immunosuppressive agents, and surgical treatment [4]. However, these treatments can only prevent the progression of the disease and promote the re-coloring of depigmented areas, but cannot completely cure vitiligo. Therefore, vitiligo's recurrence has grave impacts on the physical health, quality of life and social communication of these patients resulting in some psychological disorders, such as the development of an inferiority complex and social isolation [5,6].
The seeds of Vernonia anthelmintica (L.) Willd. (Quchong Banjiuju) are called Kaliziri in traditional Uyghur medicine, and have traditionally been used for the treatment of

The Identification of Caffeoyl Quinic Acid Derivatives in KZI by UHPLC-Q-Orbitrap-MS
The UHPLC-Q-Orbitrap-MS total ion chromatogram (TIC) of KZI is shown in Figure 1. Based on the accurate mass measurements, the key fragmental ions and the comparison with reference standards, 60 caffeoyl quinic acid derivatives were identified in KZI. The results are shown in Table 1.
Calibration curves and linearity. Calibration curves were calculated using the peak area ratio of analytes compared with the internal standard. A weighting factor of 1/X 2 was used for linearity. The method demonstrated strong linear reliability. The six analytes showed linearity with concentration ranging from 6 to 600 ng/mL in beagle plasma. The lower limit of quantification (LLOQ) for 5-CQA, 4-CQA, 1,3-diCQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA was 6 ng/mL. Accuracy and precision. The quality control (QC) sample solutions were prepared in beagle plasma for 5-CQA, 4-CQA, 1,3-diCQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA, each at 10, 100, and 400 ng/mL. Intra-day accuracy and precision were evaluated in six replicates of QC samples with different concentrations on the same day. Inter-day accuracy and precision were assessed in triplicates of QC samples with different concentrations on three days. Accuracy was calculated by comparing the mean concentration to the theoretical concentration. Precision was interpreted by the relative standard deviation (RSD). The intra-day and inter-day accuracy of six analytes at each concentration were in the range of 85-115%. The intra-day and inter-day precision of six analytes at each concentration were in the RSD range of 0-15%.
Stability. Long-term stability was evaluated in three replicates at different QC concentrations after storage at −80 • C for 20 days. Freeze-thaw stability was assessed in three replicates of different QC concentrations after exposure to three sequential freeze-thaw cycles. For each cycle, samples were frozen for more than 24 h below −80 • C, then transferred to a 4 • C environment for 2 h until completely thawed. Stability in an auto-sampler was evaluated in three replicates at different QC concentrations. Six analytes at each concentration were stable over 12 h in the auto-sampler. They were also stable after three freeze-thaw cycles and after 20 days of storage at −80 • C.  Table 3. The quantitative HPLC-MS/MS method was applied to quantify 5-CQA, 4-CQA, 1,3-diCQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA in beagle plasma samples. After the subcutaneous injection of KZI, beagle plasma exhibited quantifiable levels for all of the six analytes. As shown in Figure 4  administration of KZI, 4-CQA reached the peak plasma concentration (114.87 ng/mL); 6 h after administration, the level of 4-CQA fell to under the LLOQ. At 0.5 h after administration of KZI, 1,3-diCQA reached the peak plasma concentration (11.20 ng/mL); 2.5 h after administration, the level of 1,3-diCQA fell to under the LLOQ. At 0.25 h after the administration of KZI, 3,4-diCQA reached the peak plasma concentration (30.46 ng/mL); 2.5 h after administration, the level of 3,4-diCQA fell to under the LLOQ. At 0.25 h after administration of KZI, 3,5-diCQA reached the peak plasma concentration (19.49 ng/mL); 2 h after administration, the level of 3,5-diCQA fell to under the LLOQ. At 0.25 h after administration of KZI, 4,5-diCQA reached the peak plasma concentration (22.96 ng/mL); 2.5 h after administration, the level of 4,5-diCQA fell to under the LLOQ. Table 3. Contents of 5-CQA, 4-CQA, 1,3-diCQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA in KZI.

UHPLC-Q-Orbitrap-MS Conditions
Qualitative analysis was performed on Q Exactive Orbitrap apparatus coupled with Ultimate 3000 equipment (Thermo Fisher Scientific, Waltham, MA, USA). The column was an HSS T 3 (1.8 µm, 2.1 × 100 mm, Waters, Ireland). The column oven temperature was 40 • C. The mobile solvents were A (acetonitrile, 0.1% v/v formic acid) and B (water, 0.1% v/v formic acid) a with flow rate of 250 µL/min and in the following gradients:

Animal Experiment
Three male beagles were obtained from Xinjiang Medical University. They were fasted for 12 h before administration, but drank water freely. Animals were administrated subcutaneous KZI injection with a dose of 0.2 mL/kg. Blood was collected at 0, 0.05, 0.15, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 10 h from hind limbs veins. Urine and feces were also collected. The collected blood was centrifuged for 15 min to produce plasma samples. The plasma samples were immediately stored at −80 • C.

Treatment of Beagle Plasma Samples
Beagle plasma samples were mixed with 10 µL internal standard solution and vortexed for 10 s. Subsequently, 300 µL acetonitrile was added and the mixture was vortexed for 60 s; then, it was centrifuged for 7 min. The supernatant was collected and nitrogen dried at 40 • C. The dried samples were mixed with 100 µL methanol and sonicated for 60 s, then centrifuged for 7 min. The supernatant was collected and injected for analysis.