**1. Introduction**

Abelmoschi Corolla (AC) is derived from the dried corolla of Flos *Abelmoschus manihot* (FAM), which has the functions of eliminating dampness and heat, subduing swelling, and detoxicating [1]. It is a traditional Chinese medicine (TCM) with a long medicinal history in China [2]. The research of pharmacology showed that AC has multiple pharmacological activities, such as anti-inflammatory, antioxidant [3,4], antitumor [5], anticonvulsant, antidepressant, and neuroprotective activities [6,7], as well as therapeutic actions on renal tubular injury and diabetic nephropathy [8–10].

As the main raw material of the Chinese patent medicine Huangkui capsule, AC has significant medicinal value and a huge market demand. Market research found that commercial medicinal material of AC was often mixed with non-medicinal parts of FAM,

including calyx, stamen, and pistil. The reason for this phenomenon is that the nonmedicinal parts of FAM are not removed during the collection and processing of corolla. However, the chemical constituents of non-medicinal parts of FAM were not characterized in previous studies, so the comparative study between medicinal and non-medicinal parts of FAM was not clear-cut. The mixing of non-medicinal parts may alter the composition of AC, reduce its quality stability, and then affect its efficacy. Therefore, it is of practical value to study the chemical constituents of medicinal and non-medicinal parts of FAM and reveal the characteristics of metabolic differences.

Because it combines the separation powers of liquid chromatography with the very sensitive detection qualities of mass spectrometry, the LC-MS equipment has been frequently employed in TCM research in recent years [11]. Among them, ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS) is efficient and rapid in the determination of the molecular weight and characteristic fragment ions, by which the structure of multiple constituents in TCM can be identified quickly [12]. Hence, the characterization of the constituents in calyx, corolla, stamen, and pistil was analyzed by UFLC-Triple TOF-MS/MS. We integrated metabolic profiling and multivariate statistical analysis to separate the medicinal and nonmedicinal parts of FAM and to define their chemical markers. The strategy for comparative analysis on chemical constituents of medicinal and non-medicinal parts of FAM was shown in Figure 1. The study could determine the differential constituents of medicinal and non-medicinal parts of FAM, so as to provide basic data for standardizing the harvest and market standards of AC. Our investigation will not only contribute to the quality evaluation of AC, but also has grea<sup>t</sup> significance in the quality stability improvement of AC.

**Figure 1.** The strategy for comparative analysis on chemical constituents of medicinal and nonmedicinal parts of Flos *Abelmoschus manihot* (FAM).

### **2. Materials and Methods**

### *2.1. Chemicals and Reagents*

The standard substances of 3,4,5-trihydroxybenzoic acid, rutin, hyperin, and quercetin were purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 3,4-Dihydroxybenzoic acid was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. (Shanghai, China). Chlorogenic acid, caffeic acid, and myricetin 3 -*O*-*β*-D-glucopyranoside were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Dihydromyricetin and myricetin

were purchased from Chengdu Aifa Bio-technology Co., Ltd. (Chengdu, China). Myricetin 3- *O*-*β*-D-glucopyranoside and quercetin 3- *O*-*β*-D-robinobioside were purchased from Liangwei Bio-technology Co., Ltd. (Nanjing, China). Quercetin 7- *O*-*β*-D-glucopyranoside, gossypetin 8- *O*-*β*-D-glucuronide, and quercetin 3- *O*-(6- *O*-acetyl-*β*-D-glucopyranoside) were purchased from Nanjing Casses Pharmaceutical Technology Co., Ltd. (Nanjing, China). Isoquercetin, quercetin 3 -*O*-*β*-D-glucoside, and tiliroside were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Chengdu, China). The purities of myricetin 3 -*O*-*β*-D-glucopyranoside was above 97% and other standards were greater than 98%, tested by HPLC analysis. Formic acid, acetonitrile, and methanol of HPLC grade were purchased from Merck (Darmstadt, Germany). A Milli-Q water purification system (Millipore, Bedford, MA, USA) was applied to make deionized water.
