*2.2. Plant Materials*

Five batches of FAM samples were collected from Xinghua City (Jiangsu Province, China 32◦9817 N, 119◦9044 E) in October 2019. Each batch was carefully divided into four parts, calyx, corolla, stamen, and pistil, which were separately dried in the oven. The drying temperature was set at 50 ◦C. The samples were authenticated by Professor Xunhong Liu as the flower of *Abelmoschus manihot* (L.) Medic. of Malvaceae family and the voucher specimens were deposited in the laboratory of Chinese medicine identification, Nanjing University of Chinese Medicine. The voucher numbers of the samples were as follows: 190923CA1 −190923CA5 (calyx), 190923CO1 −190923CO5 (corolla), 190923ST1 −190923ST5 (stamen), 190923PI1 −190923PI5 (pistil).

### *2.3. UFLC-Triple TOF-MS/MS Analysis*

### 2.3.1. Preparation of Standard and Sample Solutions

A mixed standard stock solution of 18 standard substances was prepared with 70% (*v*/*v*) methanol. The diluted solutions were stored at 4 ◦C for further UFLC-Triple TOF-MS/MS analysis.

The 0.5 g of calyx, corolla, stamen, and pistil powder were properly weighed and ultrasonically extracted with 20 mL 70% (*v*/*v*) methanol for 30 min, respectively. To compensate for the weight lost during extraction, the same solvent was added after cooling to room temperature. The extract was then filtered, and the filtrate was centrifuged at 12,000 rpm/min for 10 min. Afterwards, the supernatant was filtered via a 0.22 μm membrane before UFLC-Triple TOF-MS/MS analysis.

### 2.3.2. UFLC-Triple TOF-MS/MS Conditions

The chromatographic analysis was performed on an UFLC-20AD XR system (Shimadzu, Kyoto, Japan). The separation was conducted by an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm) at 35 ◦C. The mobile phase contained 0.1% (*v*/*v*) aqueous formic acid water solution (A)–methanol:acetonitrile (1:1) (B) with the gradient elution: 0 −3 min, 2% B; 3 −10 min, 2 −15% B; 10 −14 min, 15 −18% B; 14 −20 min, 18 −21% B; 20 −30 min, 21 −23% B; 30 −45 min, 23 −27% B; 45 −50 min, 27 −40% B; 50 −52 min, 40 −80% B; 52 −54 min, 80 −95% B. The injection volume was 10 μL and the flow rate was 1 mL/min.

A Triple TOFTM 5600 System MS/MS High Resolution Quadrupole Time-of-Flight Mass Spectrometer (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization source was used for MS analysis in both positive and negative ion modes. The MS conditions were optimized as follows: the ion source temperature, 550 ◦C; the flow rate of curtain gas, 40 L/min; the flow rate of nebulization gas, 55 L/min; the flow rate of auxiliary gas, 55 L/min; the spray voltage, 4500 V in positive ion mode and -4500 V in negative ion mode; the declustering voltage, 100 V in positive ion mode and -100 V in negative ion mode. TOF MS and TOF MS/MS were scanned with the mass range of *m*/*z* 100 −2000 and 50 −1500, respectively.

### 2.3.3. Identification of the Constituents

A database of the chemical constituents of AC was formed based on previous research and the data were imported into the PeakView Software V.1.2 (AB SCIEX, Framingham, MA, USA). The chemical constituents of different parts of FAM were comprehensively characterized by comparing the retention time (*tR*), accurately measuring mass and multistage MS/MS fragmentation information with standard substances, databases, and related literatures.

### *2.4. Multivariate Statistical Analysis*

The data of UFLC-Triple TOF-MS/MS were processed by PeakView Software V.1.2 (AB SCIEX, Framingham, MA, USA) and MarkerView 1.2.1 software (AB Sciex). Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS–DA) were performed using SIMCA-P 13.0 software (Umetrics AB, Umea, Sweden). PCA was used to categorize and identify different parts of FAM. OPLS-DA was performed to differentiate medicinal part and non-medicinal parts of FAM, as well as to identify the common differential constituents that cause the differences in each group of comparison by variable importance in the projection (VIP).

### *2.5. Relative Content Comparison of Differential Constituents*

The relative contents of differential constituents in medicinal and non-medicinal parts of FAM were compared according to the peak intensities. To visualize and validate the distribution regularity of differential constituents among medicinal and non-medicinal parts of FAM, one-way analysis of variance (one-way ANOVA) was applied. Diagram of relative content comparison was charted by GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).
