Reagents, chemicals and materials
Acetonitrile (MS grade) was purchased from RCI Lab scan Ltd (Bangkok, Thailand). Analytical grade formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol and ethanol were obtained from Merck (Darmstadt, Germany). Deionized water was prepared using a Millipore MilliQ-Plus water purification system (Millipore, Bedford, MA, USA).
Reference standards of l-phenylalanine (1), catechin (2), genipin-1-gentiobioside (3), epicatechin (4), geniposide (5), leonurine (6), 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (7), cyasteron (8), corynoxeine (9), isorhynchophylline (10), isocorynoxeine (11), baicalin (12), rhynchophylline (13), oroxylinA-7-O-glucuronide (14), bacalein-6-O-beta-glucopyranoside (15), wogonoside (16) and wogonin (17) were purchased from Chengdu Preferred Biotechnology Co., Ltd (Chengdu, China). The identities of the reference standards were confirmed by mass spectrometry prior to being used. The purities of the reference standards were determined to be greater than 98 % by UPLC-DAD analysis based on peak area normalization. Reference standards of fructose, glucose and sucrose were purchased from Sigma-Aldrich. The structures of the reference standards are shown in Fig. 1.
Commercial samples (TGY1–10) were purchased from different pharmacies in several different regions of China, including Jilin, Beijing, Nanchang, Shenzhen and Hong Kong. The sample designated TGY11 evaluated in the current study was prepared in our laboratory using herbal materials purchased from the Mr & Mrs Chan Hon Yin Chinese Medicine Specialty Clinic and Good Clinical Practice Centre, Hong Kong Baptist University, China. All of the herbs used in the current study were identified and authenticated by Professor Zhong-Zhen Zhao from the School of Chinese Medicine, Hong Kong Baptist University, China. Voucher specimens were deposited at the School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China (Collection numbers A110607, 110991, A1202151, 1300942, A121017, 131110, 140102, 140112, 130906, 130603).
Sample solutions preparation
The commercial samples (TGY1–10) were ground into fine powders and passed through a 60–80 mesh filter. A small accurately weighed sample (20 mg) of each powder was then dissolved in distilled water in a 10 mL volumetric flask at room temperature. All of the resulting sample solutions were filtered through a 0.22 µm nylon-membrane filter (Millipore, Barcelona, Spain) prior to being analyzed to determine the 17 non-saccharide small molecules.
The TGY11 material prepared in our laboratory consisted of the following materials: G. elata (Tianma) (9 g), U. rhynchophylla (Gouteng) (12 g), C. officinalis (Chuan Niu Xi) (18 g), G. jasminoides (Zhizi) (9 g), S. baicalensis (Huangqin) (9 g), C. officinalis (Chuan Niu Xi) (9 g), E. ulmoides (Du Zhong) (9 g), L. japonicus (Yimucao) (9 g), T. chinensis (Sang Ji Sheng) (9 g), P. multiflorum (Ye Jiao Teng) (9 g) and P. cocos (Fuling) (9 g) [1]. All of the crude materials were powdered and extracted three times by refluxing in boiling water (1:10, w/v) for 1 h. The combined extracts were then filtered and evaporated under reduced pressure, before being freeze-dried to give the TGY11 material as a powder. This powders were then prepared according to the procedure described above for the preparation of the commercial samples.
For the analysis of the saccharides, a small accurately weighed portion (10 mg) of each sample was dissolved in 2 mL of water. Ethanol (8 mL) was then added slowly added to each aqueous solution to precipitate any macromolecular components. The resulting mixtures were held for 24 h at room temperature, before being centrifuged on a 5804 Eppendorf multi-purpose centrifuge (Eppendorf Bio Tools, Radnor, PA, USA) at 3250×g for 15 min. The supernatant was collected and evaporated to dryness to give a residue, which was dissolved in a 1:1 (v/v) mixture of ACN and water (1 mL) and filtered through a 0.22 nylon-membrane filter (Millipore). The resulting filtrates were subjected to HPLC-ELSD analysis to determine their fructose, glucose and sucrose levels. The precipitate from the centrifugation step was dried and weighed to determine the macromolecular content of each sample [17].
Standard solutions preparation
Reference standards of l-phenylalanine, catechin, genipin-1-gentiobioside, epicatechin, geniposide, leonurine, 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside, cyasteron, corynoxeine, isorhynchophylline, isocorynoxeine, rhynchophylline, baicalin, oroxylinA-7-O-glucuronide, wogonoside, baicalein and wogonin were weighed and dissolved in different volumes of methanol containing distilled water to prepare stock solutions. Samples of these stock solutions were then mixed together to prepare a mixed standard solution. The standard saccharide solutions were prepared according to the methods described in our previous publication [18]. Reference markers of fructose, glucose and sucrose were accurately weighed and dissolved in a 1:1 (v/v) mixture of ACN and water. Calibration curves were obtained by the appropriate dilution of these mixed standard solutions.
Analytical methods
Chromatographic analysis was performed on an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, a thermostat-controlled column compartment, an auto sampler and a DAD detector. Separations were conducted over an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters, Milford, CT, USA) at 40 °C with a gradient elution consisting of 0.1 % formic acid in water (mobile phase A) and 0.1 % formic acid in ACN (mobile phase B). The column was eluted with the following gradient program: 0–3 min, 2 % B; 3–9 min, 2–12 % B; 9–24 min, 12–32 % B; 24–29 min, 32–75 % B; 29–29.1 min, 75–100 % B; 29.1–32 min, 100 % B. The flow rate was set at 0.4 mL/min.
An Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream electrospray ionization (ESI) source was used to acquire the MS and MS/MS data in the positive and negative ionization modes. Data acquisition was controlled using MassHunterB.03 software (Agilent Technologies, Wilmington, USA). The operating parameters were set as follows: nebulizing gas (N2) flow rate, 8.0 L/min; nebulizing gas temperature, 300 °C; jet stream gas flow, 9 L/min; sheath gas temperature, 350 °C; nebulizer gas pressure, 45 psi; capillary voltages, 3000 V; skimmer voltage, 65 V; Oct RFV, 600 V; fragment voltage, 150 V. Mass spectra were recorded for m/z values in the range of 100–1700 with accurate mass measurements for all of the mass ions. The peak areas determined in the extracted ion chromatograms were placed into calibration curves, which were prepared by plotting the peak areas of samples containing different concentrations to calculate the non-saccharide small molecule contents contained in this decoction.
The HPLC-ELSD conditions used for the determination of the saccharides were described in our previous publication [18]. An Agilent 1100 liquid chromatograph system (Agilent Technologies, Palo Alto, CA, USA) equipped with an Alltech 2000 evaporative light-scattering detector (Grace, Deerfield, MA, USA) was used. The chromatographic separations were performed over an Asahipak NH2P-50 4E column (4.6 × 250 mm, Shodex, Tokyo, Japan) at a column temperature of 30 °C. The column was eluted with a mixture of water (mobile phase A) and ACN (mobile phase B) at a flow rate of 0.8 mL/min. The elution conditions were as follows: 0–16 min, 78 % B; 16–20 min, 78–62 % B; 20–30 min, 62–60 % B. The drift tube temperature of the ELSD was set at 120 °C, and the nitrogen flow rate was set at 3.2 L/min. The peak areas in the ELSD chromatograms were collected to calculate the concentrations of the different components. Calibration curves were generated by plotting the logarithmic values of the different peak areas against the logarithmic values of the corresponding concentrations.
Method validation
The optimum method for the quantitative analysis of the samples was validated in terms of its linearity, sensitivity, precision, accuracy and stability. Stock solutions of the mixed standards were diluted to a variety of different concentrations to allow for the construction of calibration curves. At least six concentrations of each reference standard were analyzed in triplicate. The calibration curves were constructed by plotting the peak areas versus the concentrations of the corresponding constituents. The limit of detection (LOD) and limit of quantification (LOQ) values for the optimum conditions were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. The intra- and inter-day variations were used to evaluate the precision of our newly developed method. Six independently prepared solutions of TGY11 were analyzed within 1 day to evaluate the intra-day variability of the optimum method. To evaluate the inter-day variability of this method, we examined the same sample twice a day over 3 consecutive days. Variations were expressed as relative standard deviations (RSDs) of the data, which were calculated using the following formula: \( {\text{RSD }}\left( \% \right) = \left( {{\text{standard deviation}}/{\text{mean}}} \right)\, \times 100\, \% \). A recovery test was performed to evaluate the accuracy of the optimum method by adding three different concentrations of a standard solution (i.e., low, medium and high) to TGY11, which contained known quantities of the target compounds. These samples were then analyzed in parallel using our newly established method. Each experiment was conducted in triplicate at each level. The spike recoveries were calculated using the following equation:
$$ Spike \, recovery \, \left( \% \right)= (total \, amount \, detected\,{-}\,amount \, original)/amount \, spiked \times 100 \,\% $$
The stability of the optimal method was evaluated by analyzing the TGY11 extracts over periods of 0, 2, 4, 8, 12 and 24 h. The RSDs of the peak areas of each compound were used as an indication of the stability.
