Silica gel

Sodium hydroxide (NaOH), hydrochloric acid (HCl) aluminium sulphate (Al₂(SO₄)₃·18H₂O), chromium chloride hexahydrate (CrCl₃·6H₂O), copper sulphate pentahydrate (CuSO₄·5H₂O), 10-hydroxybenzo[h]quinoline (C₁₃H₉NO, the structure is shown in scheme 1a, abbreviated as HBQ) and silica gel (30 wt% Aldrich), were purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals were of analytical grade and used without any further purification. Scheme 2 schematically illustrates the synthetic process of HBQ-M-mags (M = Al, Cr and Cu) in the interlayer spaces of mags and the detailed process is described in the following sections.
Scheme 2.

A schematic illustration of preparation of M(HBQ)_n (M = Al, Cr, Cu) in the interlayer space of mags and proposed structures of mag, H-mag, M-mag and HBQ-M-mag.

Nelumbo nucifera cv. Taikong lotus 36, the highest strain of selective lotus breed after space mutagenesis, was selected in this study (Wu et al., 2007 (link)). Seeds were sprouted by soaking in water for germination. Five days after, plants were provided with 50 cm depth pots in a greenhouse for the entire growing season. Functional leaves, petiole, rhizomes and roots were separately collected in the 8th week, 10th week, 12th week. Seeds from plants at 12, 16, 20, 24 and 28 DAF (days after fertilization) were also collected from the Taikong lotus 36 in the genetic experimental base of Wuhan University. Various materials from lotus were quick-frozen in liquid nitrogen, then stored at −80 °C for next manipulation. The fresh leaves of 45 lotus individuals (Supplement 1) were collected from the genetic experimental base of Wuhan University and stored in silica gel (Sinopharm, China).

α-Crocin (≥98%) and geniposide (≥98%) of analytical grade were purchased from Aladdin (Shanghai, China). HPLC grade acetonitrile was obtained from J&K Chemical Ltd. (Shanghai, China). Silica gel (100~200 mesh), Diatomite (filter aid, flux calcined, D50: 19.6 μm), neutral alumina (100 to 200 mesh), basic zinc carbonate (AR, 100 to 200 mesh), calcium carbonate (AR, 99%, 100 to 200 mesh) and activated carbon (AR, 200 mesh, brown) were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Aluminum polychloride (PAC) (aluminum content ≥29%, basicity = 60% to 95%, pH = 3.5~5.0) was purchased from Tianjin Dingshengxin Chemical Reagent Co. (Tianjin, China). Deionized water was further purified using a Milli-Q water-purification system from Millipore (Bedford, MA, USA).

Methanol and acetonitrile (ACN) at HPLC grade were obtained from Merck (Darmstadt, Germany). Acetic acid, anhydrous magnesium sulfate, and sodium chloride at analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Standards of thiamethoxam, acetamiprid, thiacloprid, and ethyl 6-chloropyridine-2-carboxylate (internal standard) were supplied by Aladdin (Shanghai, China). C18 and PSA were from Sepax (Suzhou, China), and silica gel was from Sinopharm. Ultrapure water (18.2 MΩ) was used in all experiments. Rape (Brassica campestris) bee pollen used for method development was collected from an apiary in Hubei, China. Commercial rape bee pollen samples used in this application were purchased from local markets. Stock solutions of standards were prepared in ACN with a concentration of 0.2 mg/mL. Working solutions of standards were prepared by further dilution with ACN. All standard solutions were stored at 4 °C until used.