Abstract
An ultra-rapid analytical method for determination of andrographolide and dehydroandrographolide in Andrographis Herba (AH) was developed by liquid chromatography with mass spectrometry (LC-MS). The sample was ultrasonically extracted with 10 mL 40% (v/v) methanol, and then purified with a C18 solid phase extraction column. The LC separation was performed on a Poroshell 120 EC-C18 column (30 × 2.1 mm, 2.7 μm) and eluted with 0.5 mmol L−1 ammonium acetate aqueous solution and acetonitrile (65:35) at a flow rate of 0.7 mL min−1, and detected by mass spectrometry (MS). The LC-MS analytical time was less than 1 min. The new developed method presented a good linearity (r > 0.9900), precision and repeatability (RSD < 2.0%). The recoveries for andrographolide and dehydroandrographolide were 93.5% (RSD = 2.2%) and 97.7% (RSD = 2.4%), respectively. The developed method was successfully applied in determination of andrographolide and dehydroandrographolide in seven batches of AH samples, and the contents of analytes in all samples were complied with the relative acceptance criteria in Chinese Pharmacopeia (>0.8%). This new developed LC-MS method is an ultra-rapid assay method for AH, which will help to improve the efficiency and reduce the cost of AH sample test.
Introduction
Andrographis Herba (AH), the dried aerial part of Andrographis paniculata (Burm. f.) Nees, is a traditional herbal medicine. It is used to clear heat, remove toxin, cool the blood, and disperse swelling [1]. Modern studies show that AH contains a lot of compounds, such as diterpene lactones, flavonoids, phenylpropanoids, iridoids, alkaloids and so on [2, 3]. Andrographolide and dehydroandrographolide, the major diterpene lactones in AH, which are believed as the important active components, showing antithrombotic, cardio-protective, antiviral, anticancer, anti-oxidant, anti-pyretic and anti-microbial activities [4–6]. Andrographolide and dehydroandrographolide are used as markers for the quality evaluation of AH in the Chinese Pharmacopoeia [1]. Several liquid chromatography (LC) methods, including LC with ultraviolet detection (LC-UV) and LC with mass spectrometry (LC-MS) [7–12], were developed for the determination of andrographolide and dehydroandrographolide in AH. However, these methods are time-consuming and require amounts of organic solvents. For example, Chen et al. (2020) used ultrasonic extraction (UE) and LC-UV for the analysis of diterpene lactones in AH, which took 95 min and cost more than 30 mL of organic solvents [7]. Fu et al. (2019) developed a method by UE and LC-MS/MS analysis, which took 37 min and consumed more than 17 mL of organic solvents [10]. Song et al. (2013) developed a method by UE and LC-TOF/MS. It consumed 9 mL of organic solvents, but test time were 45 min [12]. Therefore, it is necessary to develop a rapid and organic solvent saving analytical method for the determination of andrographolide and dehydroandrographolide in AH.
In recent years, the development of rapid LC method for the determination of bioactive components in herbal medicines has become hotspot [13, 14]. Superficially porous column, which can provide rapid separation of analytes with low back pressure, has been proved as a good choice for rapid LC analysis of herbal medicines [15–17]. MS, a selective detector, can monitor specific ions through different ion channels by selected ion mode. It has been used for rapid analysis of the difficult separated components in herbs [18–20]. Combining the advantage of superficially porous column and MS detection would provide a reasonable analytical method for rapid analysis of bioactive components in herbs. However, herbal medicine is a complicated system that usually contains a large number of components. The matrix effects often affect the quantitation of analytes in LC-MS analysis [21, 22]. Solid phase extraction (SPE) is a simple and low solvent consuming purification technology, which is easy to be operated in the common analytical labs. It has been used for the purification of investigated compounds from complex system, such as food, herbal medicine and so on [23–25]. After the SPE purification, the matrix effects of herb sample were reduced and rapid LC-MS analysis of bioactive components in herb will be achieved more easily.
In present work, an ultra-rapid assay method based on LC-MS was developed for the determination of andrographolide and dehydroandrographolide in AH, and the new method was successfully applied in seven batches AH samples.
Experimental
Chemicals and materials
LC-grade methanol and acetonitrile were obtained from KRUDE Chemical Technology Co., Ltd. (California, USA). LC-grade ammonium acetate was purchased from Aladdin (Shanghai, China). The deionized water was purified by a Milli-Q Advantage A10 water purification system (Merck KGaA, Darmstadt, Germany). Andrographolide (98%) and dehydroandrographolide (98%) were purchased from Chengdu Push Bio-technology Co. Ltd. (Chengdu, China). LC-grade gel ODS-AQ-HG (12 nm, S-50 μm) was supplied by YMC CO., LTD. (Kyoto, Japan).
Seven batches of AH samples were collected from the different herbal medicine markets in GuangDong province, China. All the samples were identified by Dr. Zhengming Qian, and the voucher specimens were deposited at the Key Laboratory of State Administration of Traditional Chinese Medicine, Dongguan, Guangdong. All crude materials were pulverized through a mill and passed through a 50-mesh sieve.
Preparation of reference compound solution
The stock reference compound solution containing andrographolide (final concentration of 4.41 mg mL−1) and dehydroandrographolide (final concentration of 4.40 mg mL−1) was prepared with methanol. The work reference compound solution was diluted from the stock reference compound solution with 40% methanol. All the reference compound solutions were kept at 4 °C before LC-MS analysis.
Preparation of sample solution
To obtain the good extraction efficiency, concentration of extraction solution, volume of extraction solution, and extraction time were optimized. The contents of andrographolide and dehydroandrographolide were used to evaluate the extraction efficiency. To get the excellent purification result, different conditions of SPE, including type of adsorbent, polarity and volume of elution solvent, were tested.
AH sample powder (100 mg) was extracted with 10 mL of 40% (v/v) methanol by ultrasonic extraction (40 kHz, 100 W) for 5.0 min. A small amount of 40% methanol solution was added to compensate the lost weight. The supernatant (1 mL) was loaded onto the SPE tube, which was filled with ODS (0.4 cm in diameter, 1.0 cm in height) which had been eluted with 2 mL 40% methanol solution before the sample loading. Subsequently, 1 mL of 40% methanol solution was employed to elute the impurities, and 4 mL of 100% methanol was employed to elute the target analytes. The target analytes solution was collected in a volumetric flask and adjusted to 5.0 mL with methanol. The solution was further filtered through a 0.22 μm nylon membrane before injected into LC-MS for analysis.
LC-MS condition
In order to obtain the ultra-rapid LC-MS condition, the optimization of MS and LC parameters was operated. The different conditions of MS, including monitoring mode, the mobile phase additive and quantitative ions were tested on a long column to obtain clean chromatography without interference peaks. The LC conditions were as follows: The sample was separated on a Waters XBridge C18 column (Waters Corporation, Massachusetts, USA) (4.6×150 mm, 5 μm) at a column temperature of 30 °C. The mobile phase was 0.5 mmol L−1 ammonium acetate aqueous solution (A) and acetonitrile (B) with a flow rate of 1 mL min−1 and used gradient elution: 0–15 min, 20–25% B; 15–30 min, 25–28%; 30–60 min, 28–40% B. The split ratio was 1:1. The injection volume was 2 µL. In addition, the different LC separation conditions were tested to get a rapid LC separation, such as column types, mobile phase compositions, column temperatures, and flow rates.
The optimized LC-MS conditions were as follows: The LC separation was carried out on an Agilent 1260 LC system (Agilent Technologies, Inc., California, USA), consisting of a binary pump, a thermostatic column compartment and an auto-sampler. Poroshell 120 EC-C18 column (Agilent Technologies, Inc., California, USA) (30 × 2.1 mm, 2.7 μm) was used for separation of the AH sample solution. The mobile phase consisted of 0.5 mmol L−1 ammonium acetate aqueous solution and acetonitrile (65:35) at a flow rate of 0.7 mL min−1. The column temperature was 40 °C and the injection volume was 2 μL.
Analytes were detected by an Agilent 6130 single quadrupole MS (Agilent Technologies, Inc., California, USA) equipped with an electrospray ionization (ESI) source in positive ionization and selected ion monitoring (SIM) mode. The monitored ions were m/z 368 for andrographolide and m/z 665 for dehydroandrographolide. Other conditions were as follows: drying gas (N2) flow rate, 10.0 L min−1; temperature, 300 °C; nebulizer, 30 psig; fragmentor, 70 V; capillary, 3000 V.
Additionally, we recommend performing column cleaning after every 30 sample injections by implementing a washing process. Mobile phase A is 0.5 mmol L−1 ammonium acetate aqueous solution and acetonitrile (65:35) and mobile phase B is acetonitrile. Changes in gradient: 0–5 min, 0% B; 5–10 min, 0%–100% B; 10–20 min, 100% B; 20–25 min, 100%–0% B; 25–30 min, 0% B. The flow rate was 0.7 mL min−1.
Method validation
According to the Chinese Pharmacopeia relative chapter, the linearity, limit of quantification (LOQ), precision, accuracy, and stability of the method were validated.
Linearity and LOQ
A series of concentrations of andrographolide (5.5–110.3 μg mL−1) and dehydroandrographolide (5.5–110.0 μg mL−1) were prepared based on the contents of them in the AH herbs [7–12]. The calibration curves were obtained by plotting peak areas versus the concentrations of reference compounds. The LOQ was determined by analyzing the reference compound solution and calculated as analyte concentration giving signal-to-noise ratios (S/N) of approximately 10.
Precision and repeatability
Precision of the method was evaluated by analyzing reference compound solution and sample solution in six replicates within one day. The repeatability was determined by analyzing sample S6 in six replicates according to the method mentioned above. The relative standard deviation (RSD, %) was used as a measure of precision and repeatability.
Recovery
The recovery was used to evaluate the accuracy of the developed assay. A known amount of the two analytes were added into sample S6. The mixture was extracted and analyzed by the optimized method. Six replicates were performed for the test. The recovery rate was calculated as 100% × (found amount – original amount)/spiked amount.
Stability
The freshly prepared sample S6 solution was stored at room temperature and analyzed at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The variation expressed as RSD% was used to evaluate the stability.
Result and discussion
Optimization of extraction conditions
Andrographolide and dehydroandrographolide were usually extracted by ultrasonic wave for more than 20 min. To improve the extraction conditions, the concentration of methanol aqueous solution, the volume of extraction solution and the extraction time were optimized. Methanol was selected as the extraction solvent based on previous studies. Three concentrations of methanol (15%, 40%, and 65%) (%, v/v) were evaluated. The results (Fig. 1A) show that 40% and 65% methanol aqueous solution present better extraction of analytes than 15%. The 40% ethanol aqueous solution was selected due to less organic solvent cost. Furthermore, the extraction volumes (2.5, 5, 10, and 15 mL) were compared. The results (Fig. 1B) showed that extraction yields by 10 and 15 mL were higher. 10 mL was used for less solvent consuming. Extraction time is also an important factor. Figure 1C showed that the extraction efficiency of different extraction times (5, 10, 20, and 30 min) was similar. Therefore, 5 min was used in experiment.
Effects of different parameters on the extraction efficiency of andrographolide and dehydroandrographolide. (A) concentration of methanol; (B) volume of extraction solvent; (C) extraction time
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01156
In order to reduce the matrix effect of AH sample solution to two analytes, the sample solution was purified by SPE before LC-MS analysis. The different conditions of SPE were tested to obtain a good experiment condition. Three adsorbents, including diatomaceous earth, aluminium oxide and ODS were compared. The ODS showed the best retention of andrographolide and dehydroandrographolide. It was used as the absorbent in current experiment. The polarity and volume of the impurity removal solution were critical for removing impurities and retaining the target components. The extraction solution 40% methanol was selected as the washing solvent, due to the good elution of impurities and retention of two analytes [7, 8, 10, 11, 26]. The volumes (1, 2, 3, 4 and 5 mL) of washing solvent were evaluated and 1 mL was found enough to remove the polar impurities. The methanol was used to elute two analytes and 4 mL methanol was proved to elute the target analytes completely. The LC analysis results of the sample solution without SPE purification and with SPE purification were presented in Fig. S1.
Optimization of LC-MS conditions
In the current study, the single quadrupole MS was used for the detection. By comparison of MS response of two analytes in the positive ion and negative ion mode, it was found that two analytes showed better response value in positive ion mode (Fig. S2). In addition, [M + NH4] + (m/z 368) for andrographolide and [2M + H] + (m/z 665) for dehydroandrographolide were selected for the quantitative analysis due to higher responsiveness (Fig. S3). Two mobile phase additives (acetic acid and ammonium acetate) were compared. The specificity and response value of two target components are better in the ammonium acetate aqueous solution - acetonitrile than acetic acid aqueous solution - acetonitrile. There were no interference peaks to the two analytes (Fig. S4). Thus, the ammonium acetate aqueous solution - acetonitrile was selected as the mobile phase additive.
After the confirmation of the MS condition, the LC condition was optimized. Andrographolide and dehydroandrographolide were usually separated on common C18 columns more than 10 min. To shorten the separation time, a type of rapid separation column, superficially porous column-poroshell column was employed in this study. Two different compositions of acetonitrile and 0.5 mmol L−1 ammonium acetate aqueous solution (30:70 and 35:65) were tested as mobile phase. The mobile phase consisting of 35% acetonitrile and 65% 0.5 mmol L−1 ammonium acetate aqueous solution was chosen for its suitable resolution and short separation time for the two analytes (Fig. S5). Meanwhile, three column temperatures (35, 40, and 45 °C) were compared, and the results showed that the separation time and resolution at three temperatures were similar and 40 °C was used in the current experiment (Fig. S6).
Method validation
The regression equation for andrographolide was y = 20694x + 919360 (r = 0.9916) in the test range of 5.5–110.3 μg mL−1, for dehydroandrographolide was y = 36179x + 116111 (r = 0.9939) in the test range of 5.5–110.0 μg mL−1. The LOQs for andrographolide and dehydroandrographolide were 0.11 μg mL−1 and 0.44 μg mL−1, respectively.
The RSDs of precision (n = 6) for andrographolide and dehydroandrographolide in the reference compound solution were 1.6% and 1.9%, respectively; The RSDs of precision (n = 6) for andrographolide and dehydroandrographolide in the sample solution were 0.55% and 1.4%, respectively. The RSDs of repeatability (n = 6) for andrographolide and dehydroandrographolide were 1.5% and 1.0%, respectively. The recoveries of andrographolide and dehydroandrographolide were 93.5% and 97.7% (Table 1), respectively. The RSDs of stability (24 h) for andrographolide and dehydroandrographolide were 2.7% and 2.8%, respectively. Thus, the developed LC-MS method was accurate and stable for determination of andrographolide and dehydroandrographolide in AH.
The recoveries of the developed LC-MS method
| Analytes | Sample | Original | Spiked | Found | Recovery | Average recovery | RSD |
| (mg) | (mg) | (mg) | (%) | (%) | (%) | ||
| Andrographolide | 1 | 0.4511 | 0.6009 | 1.0061 | 92.36 | 93.46 | 2.2 |
| 2 | 0.4553 | 0.6009 | 1.0227 | 94.43 | |||
| 3 | 0.4476 | 0.6009 | 0.9884 | 90.01 | |||
| 4 | 0.4553 | 0.6009 | 1.0314 | 95.87 | |||
| 5 | 0.4538 | 0.6009 | 1.0195 | 94.14 | |||
| 6 | 0.4511 | 0.6009 | 1.0157 | 93.96 | |||
| Dehydroandrographolide | 1 | 0.7267 | 0.8489 | 1.5673 | 99.03 | 97.73 | 2.4 |
| 2 | 0.7334 | 0.8489 | 1.5692 | 98.47 | |||
| 3 | 0.7210 | 0.8489 | 1.5136 | 93.37 | |||
| 4 | 0.7334 | 0.8489 | 1.5782 | 99.53 | |||
| 5 | 0.7310 | 0.8489 | 1.5534 | 96.88 | |||
| 6 | 0.7267 | 0.8489 | 1.5679 | 99.10 |
Analysis of AH sample
The developed method was applied in the determination of andrographolide and dehydroandrographolide in seven batches of AH samples. The representative chromatograms were shown in Fig. 2 and the determination results were listed in Table 2. The total contents of andrographolide and dehydroandrographolide in AH were 0.90~1.82%. The seven AH samples were all complied with the relative acceptance criteria in Chinese Pharmacopeia (≥0.80%). The dehydroandrographolide was the major component in AH, the content of dehydroandrographolide was higher than andrographolide in all samples. These results were consistent with the literatures [7–9].
LC-MS chromatograms of reference compounds and AH sample. (1) andrographolide; (2) dehydroandrographolide
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01156
The contents of andrographolide and dehydroandrographolide in AH samples
| No. | Content/% | ||
| andrographolide | dehydroandrographolide | Total | |
| S1 | 0.34 | 0.57 | 0.91 |
| S2 | 0.46 | 0.56 | 1.02 |
| S3 | 0.41 | 0.56 | 0.97 |
| S4 | 0.37 | 0.53 | 0.90 |
| S5 | 0.63 | 0.81 | 1.44 |
| S6 | 0.76 | 1.06 | 1.82 |
| S7 | 0.69 | 0.91 | 1.60 |
Comparisons of the developed LC method and reported ones
The comparisons of the current method with reported methods were summarized in Table 3. The total analysis time of reported methods, including sample extraction and LC separation, are more than 35 min. For example, method 1 took 30 min for the UE extraction and 65 min for the LC separation. In order to reduce the analytical time, LC-MS method was developed (method 2, 4, 6), which only cost 7–15 min in LC separation. However, the sample extraction time still needed 30 min. Method 3, which used a vacuum assisted extraction method, shortened the extraction time to 16 min, but the LC separation still cost 25 min. In current method, the total analytical time, including sample extraction (5 min) and LC-MS separation (1 min), is only 6 min.
Comparison of the developed method and reported methods for the analysis of andrographolide and dehydroandrographolide in A. Herba
| No. | Analytes | Sample preparation | Sample detection | Total timeg (min) | Total solvent consumption (mL) | Ref. | |||||
| Extraction method | Solvent consumed | Time consumed (min) | Other steps | Method | Solvent consumedf | Time consumed (min) | |||||
| 1 | APa DAPb | UEd | methanol 10 mL | 30 | filtration | LC-UV | acetonitrile 20.68 mL | 65 | 95 | 30.68 | [7] |
| 2 OCsc | |||||||||||
| 2 | AP DAP 1 OCs | UE | methanol 22.5 mL | 30 | centrifugment, filtration | LC–DAD–ESI/MS | acetonitrile 6.85 mL | 15 | 45 | 29.35 | [8] |
| 3 | AP DAP | VAEe | ethanol 6 mL | 16 | filtration | LC-UV | acetonitrile 11.25 mL | 25 | 41 | 17.25 | [9] |
| 4 | AP DAP 1 OCs | UE | methanol 15 mL | 30 | filtration | LC-ESI-MS/MS | methanol 1.56 mL | 7 | 37 | 16.56 | [10] |
| 5 | AP DAP 5 OCs | UE | methanol 100 mL | 75 | / | LC-UV | acetonitrile 11.24 mL | 35 | 110 | 111.24 | [11] |
| 6 | AP DAP 1 OCs | UE | Ethanol 7 mL | 30 | filtration | LC-TOF/MS | acetonitrile 1.92 mL | 15 | 45 | 8.92 | [12] |
| 7 | AP DAP | UE | methanol 10.2 mL | 5 | SPE purification, filtration | LC-MS | acetonitrile 0.25 mL | 1 | >6 | 10.45 | This work |
a AP: andrographolide; b DAP: dehydroandrographolide; c OCs: other components; d UE: ultrasonic extraction; e VAE: vacuum assisted extraction; f The mobile phase additives (<0.1 mL) were not reflected; g Other steps were not included in the total time
Additionally, the organic solvents, such as acetonitrile, methanol and ethanol, were always used in sample extraction and LC separation. The organic solvent consumption of the previously reported methods was in the range of 9–112 mL. For example, the method 1 consumes 10 mL methanol in the extraction and 20.7 mL acetonitrile in the LC separation. In method 2, the LC-MS only consumes 6.85 mL acetonitrile due to the rapid LC separation, but 22.5 mL methanol was used in sample extraction. Method 3 consumes 6 mL ethanol in the extraction owing to the high extraction efficiency of vacuum assisted extraction (VAE), but 11.25 mL acetonitrile was used in LC separation. Although method 6 only consumed 9 mL organic solvent, LC-TOF/MS is a very expensive instrument, which is limited to be applied in many labs. In comparison, the developed method in this study only consumes about 10 mL organic solvents for sample extraction and LC separation.
Conclusion
In this study, an ultra-rapid assay method for the determination of andrographolide and dehydroandrographolide in AH based on SPE and LC-MS was established. It is an improved analytical method for the determination of andrographolide and dehydroandrographolide in AH and related products. The SPE combined with LC-MS was a good strategy for rapid determination of components in herbs.
Conflict of interest
The authors declare no potential conflicts of interest with respect to the research, authorship, and or publication of this article.
Acknowledgements
This study was financially supported by the Key Laboratory of Guangdong Drug Administration (2021ZDB05).
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2023.01156.
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