Phytochemical Characterization for Quality Control of Phyllostachys pubescens Leaves Using High-Performance Liquid Chromatography Coupled with Diode Array Detector and Tandem Mass Detector

Phyllostachys pubescens leaves are cultivated in a number of Asian countries and have been used for antipyretic and diuretic effects since ancient times, especially in Korea. The purpose of this study was to develop and validate of analytical method for quality control of P. pubescens leaves using high-performance liquid chromatography with diode array detector (HPLC–DAD) and liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection. HPLC–DAD analysis was conducted with a Gemini C18 column, and distilled water–acetonitrile (both with 0.1% (v/v) formic acid) mobile-phase system. For the LC–MS/MS analysis, all markers were separated with a Waters ACQUITY UPLC BEH C18 column and gradient flow system of distilled water containing 0.1% (v/v) formic acid and 5 mM ammonium formate–acetonitrile. In both method, major components were detected at 2.13–11.63 mg/g (HPLC–DAD) and 0.12–19.20 mg/g (LC–MS/MS). These methods were validated with respect to linearity (coefficient of determination >0.99), recovery (95.22–118.81%), accuracy (90.52–116.96), and precision (<4.0%), and were successfully applied for the quantitative analysis of P. pubescens leaves.

Studies on the biological activity of bamboo leaves have identified anticancer, antiinflammatory, antibacterial, antiviral, diuretic, and antiobesity effects [2,3,10]. Among the many activities of bamboo species leaves, those of P. pubescens have been reported to have antifungal, antiobesity, antioxidant, and anticoagulant effects [11][12][13]. Vinpocetine, isolated from leaves of P. pubescens, has also been reported to prevent osteoblast apoptosis and osteonecrosis of the femoral head [14]. We recently investigated the effect of extracts from P. pubescens leaves on SRD5A2 gene expression in human prostate cell lines and an animal model of testosterone-induced benign prostatic hyperplasia [15].
A range of analysis methods has been reported for phytochemical profiling and quantification of P. pubescens leaves; these include methods based on high-performance liquid chromatography (HPLC) with ultraviolet or diode array detection (DAD), thin-layer chromatography, liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy, and gas chromatography-mass spectrometry (GC-MS) [1][2][3][4][5][6]8,16,17]. However, no simultaneous assay for the analysis of flavonoids and phenylpropanoids in P. pubescens leaves using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated.
GC, GC-MS, HPLC, and LC-MS systems have long been used for qualitative and quantitative analysis of traditional Chinese medicine (TCM), traditional Korean medicine (TKM), and Kampo medicine (KM) such as herbs or herbal products. Although HPLC systems are the most commonly used analytical instrument for quantitative analysis of TCMs, LC-MS systems can be used to rapidly and accurately detect large numbers of chemicals, and the latter approach is being used more frequently because of the complexity of TCM and various characteristics of phytochemicals [18,19].
The purpose of the present study was development and validation of two rapid, accurate, and sensitive quantification methods (HPLC-DAD and LC-MS/MS) to determine the six marker components (chlorogenic acid, isoorientin, orientin, isovitexin, vitexin, and p-coumaric acid) for quality control of P. pubescens leaves.

Method Validation of the Developed HPLC Assay
The system suitability parameters capacity factor (k ), selectivity (α), theoretical plate number (N), resolution (Rs), and tailing factor (Tf ) were tested to assess the stability of the measurements and operation of the HPLC system; as shown in Table S2, the corresponding values were 2.94-4.06, 1.03-1.19, 399752-722500, 2.62-14.09, and 1.051.10. As shown in Table 1, the coefficient of determination (r 2 ) values of the five marker components were 1.0000, indicating excellent linearity. By using Equations (1) and (2) (Section 4.6), limit of detection (LOD) and limit of quantification (LOQ) values were calculated to be 0.03-0.10 µg/mL and 0.09-0.29 µg/mL, respectively ( Table 1). The recovery (%) of the five marker analytes was 95.22-101.29% from Equation (3) ( Table 2), and precision of relative standard deviation (RSD, %) 1.50% was calculated by Equation (4) (Tables 3, S3 and S4). These data confirmed that the HPLC-DAD method developed in this study can be used to rapidly and simultaneously analyze the five marker components in P. pubescens leaves extract. a y and x represent the peak area and concentration of each marker analyte, respectively. b LOD means the limit of detection. c LOQ means the limit of quantification.  Table 3. Precision of the developed HPLC analysis method using 5 markers.

Quantification of the Five Markers in P. pubescens Leaves Samples by HPLC-DAD Analysis
The optimized HPLC-DAD assay was successfully applied to simultaneous quantitation for quality control of P. pubescens leaves. Table 4 shows the content of each marker in samples of freeze-dried P. pubescens leaves; the concentrations of the five marker components were determined to be 1.71-11.63 mg/g. Table 4. Quantitation of the five marker compounds in samples of P. pubescens leaves by HPLC-DAD (n = 3).

Batch 1 Batch 2 Batch 3
Mean Optimal analysis conditions were screened for quantitative analysis of P. pubescens leaves with the LC-MS/MS system (Waters, Milford, MA, USA) combined with a Waters ACQUITY ultra-performance liquid chromatography (UPLC) I-Class system and Xevo TQ-XS tandem quadrupole mass spectrometer. In this system, six markers were separated and quantified with an ACQUITY UPLC BEH C 18 column (2.1 mm × 100 mm, 1.7 µm, Waters, Milford, MA, USA) maintained at 45 • C under gradient elution conditions of distilled water, containing 0.1% (v/v) formic acid and 5 mM ammonium formate, and acetonitrile as a mobile phase. Simultaneous analysis using the multiple reaction monitoring (MRM) mode (Tables 5 and S5)   For the simultaneous analysis, P. pubescens leaves using the six markers with the established LC-MS/MS method, the MRM transition, the precursor ion (Q1), and product ion (Q3) of each marker were set, as shown in Table 5. The Q3 peak of chlorogenic acid was detected at m/z 191.0 as [quinic acid−H] − , formed by the loss of the caffeoyl group in Q1 [20]. The Q3 ion peak for p-coumaric acid was detected at m/z 147.0 as [M + H − H 2 O] + , with the loss of a mass of a water molecule from the Q1 peak [21]. The Q3 peaks of 6-C-glycosides, orientin, and vitexin, and 8-C-glycosides, isoorientin, and isovitexin were detected at m/z 327.1, 311.1, 327.1, and 311.1, respectively. All the C-glycosides were detected as 0,2 X − ([M-H-C 4 H 8 O 4 ] − ) ( Figure S2) forms in which water molecule was removed from the Q1 peak [22,23].  Table 6). The recovery of the markers was calculated from Equation (3) to be 96.56-118.81% (Table 7), and an RSD of <4.0% for precision was established by using Equation (4) ( Table 8). These results validate the analytical method developed for quality control of P. pubescens leaves using LC-MS/MS.

Quantification of the Six Marker Components in P. pubescens Leaves by LC-MS/MS MRM Mode
The LC-MS/MS analysis method developed and validated for quality assessment of P. pubescens leaves using the six marker analytes was successfully applied to the analysis of 80% ethanol extract. Two phenylpropanoids (chlorogenic acid and p-coumaric acid) and four C-glycosides (isoorientin, orientin, vitexin, and isovitexin) were eluted at 10.17, 14.10, 18.27, 18.60, 20.56, and 21.12 min, respectively (Figures 2 and S3). The amounts of marker substances in P. pubescens leaves are shown in Table 9, which were detected in concentrations of 0.12-19.20 mg/g.
Among the various phytochemicals, we selected 10 components (chlorogenic acid, caffeic acid, isoorientin, orientin, isovitexin, vitexin, p-coumaric acid, ferulic acid, scopoletin, and tricin) HPLC-DAD analysis and attempted to analyze them using the water-acetonitrile (containing both 0.1% (v/v) formic acid) mobile-phase system. As a result, only the five components (chlorogenic acid, isoorientin, orientin, isovitexin, and p-coumaric acid) were detected ( Figure S3), and these components were selected as markers for the development of a simultaneous analysis method for quality control of P. pubescens leaves using HPLC-DAD. Optimal analysis conditions were developed in analysis systems using the selected markers, and the developed method was verified with respect to the linearity, LOD, LOQ, accuracy, and precision. By using the developed and validated HPLC-DAD analytic method, all markers were eluted within 20.0 min (Figure 1). It was found that isoorientin was the most abundant in the established assay. In the analysis of P. pubescens leaves using HPLC reported by Wang et al. [9] and Jin et al. [24], flavone C-glycoside, isoorientin, was detected the most. These results show analysis results equivalent to those of our study.
Simultaneous quantitative analysis for quality control of P. pubescens leaves was performed using LC-MS/MS along with HPLC analysis. In order to select a marker analyte, LC-MS/MS MRM analysis was attempted on eight components (chlorogenic acid, caffeic acid, isoorientin, orientin, isovitexin, vitexin, p-coumaric acid, and ferulic acid) among various phytochemicals reported in P. pubescens leaves [1][2][3][4][5][6][7][8][9]. Six components (chlorogenic acid, p-coumaric acid, isoorientin, orientin, vitexin, and isovitexin) of them were detected in the P. pubescens leave sample ( Figure S4), and these were selected as marker analytes for simultaneous analysis for quality control of P. pubescens leaves. An LC-MS/MS MRM method for the simultaneous quantification of P. pubescens leaves was developed using the selected markers, and this method was verified through linearity, LOD, LOQ, accuracy, and precision. Under the established LC-MS/MS MRM method, all markers were eluted within 22.0 min (Figure 2). Few studies have been reported on quantitative methods using LC-MS/MS. In the study reported by Wang et al., the LC-MS profile analysis was reported for four flavone C-glycoside components (isoorientin, orientin, vitexin, and isovitexin) [9], but no studies were conducted on quantitation and method validation. As a result of simultaneous analysis of P. pubescens leaves in the assay established in this study, isovitexin and isoorientin were found to have high concentrations of 19.20 mg/g and 9.33 mg/g, respectively.
Based on the above data, our study may be usefully applied to quality control of P. pubescens leaves in further studies.

Plant Materials and Preparation of 80% Ethanol Extract of P. pubescens Leaves
Dried Chinese P. pubescens leaves (2020PPL) that are more than 3 years old were collected, dried naturally. The extract (production number: KOC-ZY-20191008) was processed by Zhenjiang KOC Biotech Co., Ltd. (Zhenjiang, China), a company specializing in herbal extracts. For the extraction process, the dried sample was extracted using 80% ethanol at 80 • C for 3 h and then filtered using a 100 mesh (150-µm) sieve. The extract was concentrated at 60 • C for 5 h under the pressure of 0.08-0.10 MPa and then dried with a microwave dryer, to obtain a powder sample in a yield of 10.0%.

HPLC-DAD Analytical Conditions
A Shimadzu Prominence LC-20A (Kyoto, Japan) linked to an SPD-M20A DAD was used as the HPLC system for simultaneous quantification of marker analytes in P. pubescens leaves. The system was controlled by LabSolution software (version 5.53, SP3, Kyoto, Japan). Analytical conditions such as analytical column, mobile phase, and gradient elution conditions for simultaneous quantification of marker components were determined according to a previous protocol [15]. The markers were quantified by measuring absorbance at 310 nm for p-coumaric acid, 325 nm for chlorogenic acid, 335 nm for isovitexin, and 350 nm for isoorientin and orientin using a DAD that simultaneously scanned from 190 to 400 nm. Details of the operating conditions are summarized in Table S1.

LC-MS/MS Analytical Conditions
The LC-MS/MS system consisted of an ACQUITY UPLC system (Waters, Milford, MA, USA) fitted with two pumps, a column oven, an auto-sampler, and a Xevo TQ-XS MS system coupled to an electrospray ionization source. The system was controlled by Waters MassLynx v4.2 software (Waters, Milford, MA, USA). The operating conditions used for UPLC and MS for quantitative analysis of P. pubescens leaves are summarized in Table S5, and conditions for the LC-MS/MS MRM analysis are shown in Table 5.

Preparation of Standard Solutions of Marker Analytes and Sample Solution
Marker analytes were accurately weighed and dissolved in methanol or methanol-DMSO (1:1) to a concentration of about 1.0 mg/mL and used as a standard solution. Each prepared standard stock solution was degassed in a sonicator and filtered through a 0.2 µm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA). All stock solutions were stored in a refrigerator until the HPLC or LC-MS/MS analysis.
A sample solution for simultaneous analysis for quality control of P. pubescens leaves was prepared by dissolving 80% ethanol extract of P. pubescens leaves in 70% methanol at a concentration of 10 mg/mL. The solution was prepared by ultrasonic extraction for 60 min and then filtered through a 0.2 µm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA). For the LC-MS/MS analysis, the prepared sample solution was diluted 10-fold prior to use.

Method Validation of Developed HPLC-DAD Assay
The developed HPLC-DAD analytical method was validated by testing linearity, range, LOD, LOQ, recovery, and precision. The linearity was established by determining the r 2 value from the regression equation of the calibration curve prepared from a range of concentrations of each marker analyte: 0.31-20.00 µg/mL for chlorogenic acid, orientin, isovitexin, and p-coumaric acid and 0.78-50.00 µg/mL for isoorientin. LOD and LOQ were calculated from Equations (1) and (2) as follows: where σ and S are the standard deviation (SD) of the y-intercept and the slope of the calibration curve, respectively. Recovery assays were used to establish accuracy. Thus, a known amount (low, medium, and high) of five markers was spiked into a sample and the recovered amount was calculated from Equation (3) as follows: Precision was assessed with respect to repeatability, intra-day precision (within one day), and inter-day precision (successive three days), and reported as the RSD (%). Repeatability was evaluated by RSD (%) of retention time and peak area of each marker after six repeated measurements using a mixed standard solution. Intra-day and inter-day precision were also assessed by RSD (%) values. The RSD was calculated by Equation (4) as follows:

Method Validation of the Developed LC-MS/MS MRM Assay
The LC-MS/MS MRM method was validated with respect to linearity, range, LOD, LOQ, accuracy, and precision, as described for the HPLC method (Section 4.6). The linearity was determined by the r 2 of the calibration curves of each analyte prepared at different concentrations: 75.00-1200.00 ng/mL for chlorogenic acid and p-coumaric acid, 750.00-12,000.00 ng/mL for isoorientin, 200.00-3200.00 ng/mL for orientin and isovitexin, and 40.00-640.00 ng/mL for vitexin. LOD, LOQ, recovery, and precision were calculated and evaluated based on Equations (1)-(4), respectively.

Statistical Analysis
Data were presented as mean, SD, and RSD (%) by using Microsoft Excel 2019 software (Microsoft Co., Redmond, WA, USA).

Conclusions
We developed a method for the simultaneous analysis of major marker components in P. pubescens leaves based on widely used and convenient HPLC-DAD instrumentation and by using the fast, accurate, and sensitive the LC-MS/MS MRM method. Both methods were validated with respect to linearity, LOD, LOQ, recovery, and precision and met all required standards. Furthermore, the developed methods were successfully applied to the analysis of samples of P. pubescens leaves. Therefore, the analytical method described herein can be applied for quality control of P. pubescens leaves. In particular, the LC-MS/MS method will be useful for analyzing complex TCM, TKM, and KM containing P. pubescens leaves and for the study of pharmacokinetics and bioavailability using human plasma.