Characteristic HPLC–MS Profile Of Cistanche Deserticola Formula Granules
Oct 13, 2025
Authors: Mao Dan; Yu Hong; Mao Xiuhong; Hu Qing; Ji Shen*
Affiliation: Shanghai Institute for Food and Drug Control; NMPA Key Laboratory for Quality Control of Traditional Chinese Medicines, Shanghai 201203, China
Abstract
A characteristic chromatographic profiling method for Cistanche deserticola formula granules was established based on HPLC–MS. Using authenticated Cistanche deserticola decoction pieces and an echinacoside reference substance as references, ten batches of samples were extracted directly with 50% methanol and analyzed by HPLC. Separation was performed on a Phenomenex Luna C18 column (4.6 mm × 250 mm, 5 μm) with a gradient of acetonitrile–0.1% formic acid at 1.0 mL/min, 30°C, and UV detection at 330 nm. A reference characteristic chromatogram with nine common peaks was constructed, among which five peaks were identified by LC/Q‑TOF‑MS. The ten batches showed similarity ≥ 0.981. Precision, stability, and repeatability metrics of the characteristic profile were satisfactory. The method is simple, effectively identifies Cistanche deserticola formula granules, and can be used for quality evaluation.
Keywords: Cistanche deserticola; formula granules; characteristic chromatogram; echinacoside; verbascoside (acteoside); HPLC; LC–Q‑TOF‑MS; cistanche benefits; best cistanche for sale
Introduction of Cistanche
Cistanche (Rou Cong Rong) is a renowned tonic traditional Chinese medicine (TCM) with kidney‑tonifying, essence‑nourishing, and laxative effects. It was first recorded in Shennong's Classic of Materia Medica and listed as a superior grade drug. The 2020 edition of the Pharmacopoeia of the People's Republic of China (Part I) defines the authentic sources as the dried, scaly, fleshy stems of Cistanche deserticola Y.C. Ma or Cistanche tubulosa (Schenk) Wight, with processing methods including the raw product and the wine‑processed product. Major chemical components include phenylethanoid glycosides (PhGs), iridoids, lignans, and sugars, underpinning pharmacological activities such as neuroprotection, anti‑aging, hepatoprotection, and laxation; clinical application prospects are broad. Among these, PhGs are the most active constituents.
Cistanche deserticola formula granules are single‑herb granules prepared from processed C. deserticola using modern manufacturing techniques for use in clinical prescriptions. There is currently no national standard for their quality control. Prior studies reported content determination methods or fingerprinting for C. tubulosa formula granules, but no literature on characteristic chromatograms specifically for C. deserticola formula granules. In this study, ten batches were collected to establish, for the first time, an HPLC characteristic profile for C. deserticola formula granules, with five peaks identified by LC–MS. This profile provides an effective and scientific basis for rigorous quality control of C. deserticola formula granules.
Experimental of cistanche
1.1 Instruments and reagents
Agilent 1100 HPLC with UV–Vis detector and ChemStation (Agilent, USA)
Agilent 1290 Infinity HPLC and Agilent 6530 Q‑TOF MS with ESI source (Agilent, USA)
Sartorius electronic balance (Sartorius Scientific Instruments, Beijing, China)
Elmasonic P ultrasonic cleaner (Elma, Germany)
Reference decoction pieces of Cistanche deserticola, and reference substances of echinacoside and verbascoside (acteoside) were purchased from the National Institutes for Food and Drug Control (batch Nos. 121101‑201603, 111670‑201907, 111530‑201914). Cistanoside A, tubuloside A, and isoverbascoside were obtained from Shanghai Tauto Biotech (batch Nos. 21040621, 20061231, 21040824). Acetonitrile (HPLC grade) was from Merck; other reagents were analytical grade (Shanghai Lingfeng Chemical Reagents). Ten batches of C. deserticola formula granules (PFKL1–PFKL10), three batches of C. deserticola decoction pieces (YP1–YP3), and three batches of C. deserticola standard decoctions (BZTJ1–BZTJ3) were supplied by Shanghai Wanshicheng Pharmaceutical Co., Ltd.
1.2 Preparation of solutions
1.2.1 Reference solutions
Reference herb solution: Weigh 1 g of authenticated C. deserticola decoction pieces into a stoppered conical flask, add 25 mL of 50% methanol, sonicate (250 W, 35 kHz) for 30 min, cool, mix, and centrifuge. Use the supernatant as the reference herb solution.
Reference substance solution: Prepare echinacoside at 0.2 mg/mL in 50% methanol.
1.2.2 Test solution
Grind the sample; accurately weigh about 1 g into a 100 mL amber volumetric flask. Add 50 mL of 50% methanol, stopper, shake, weigh, soak for 30 min, and sonicate for 40 min (250 W, 35 kHz). Allow to cool, reweigh, and make up the lost weight with 50% methanol. Mix, stand, filter, and collect the subsequent filtrate for analysis.
1.3 Instrumental conditions
Chromatographic conditions: Phenomenex Luna C18 (250 mm × 4.6 mm, 5 μm); mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid in water; gradient elution: 0–5 min, 12% A; 5–15 min, 12%→20% A; 15–30 min, 20%→30% A. Flow rate 1.0 mL/min; detection wavelength 330 nm; column temperature 30°C; injection volume 10 μL. The theoretical plates, calculated on the echinacoside peak, should be ≥ 5000.
Note: The original text lists "15–30 min, 20%→30% A" twice; this is interpreted as a single step.
Mass spectrometric conditions: ESI in positive‑ion mode; scan range m/z 50–1200. Source settings: nebulizer (N2) 35 psi; drying gas (N2) 15 L/min at 200°C; sheath gas (N2) 12 L/min at 350°C; capillary 3.5 kV; fragmentor 125 V; MS/MS collision energies (CID) at 10, 20, and 40 eV.
Results and discussion
2.1 Sample pretreatment and detection wavelength
Extraction solvents (methanol, 75% methanol, 50% methanol, 25% methanol), extraction modes (ultrasonication, reflux), and times (10, 20, 40, 60 min) were compared. The optimal pretreatment was 50% methanol with 40 min ultrasonication. As PhGs in C. deserticola show maximum UV absorption at 330 nm, 330 nm was selected for detection.
2.2 Selection of chromatographic conditions
2.2.1 Columns
Zorbax SB‑C18 (250 mm × 4.6 mm, 5 μm), Kromasil 100‑5‑C18 (250 mm × 4.6 mm, 5 μm), and Phenomenex Luna C18 (250 mm × 4.6 mm, 5 μm) provided adequate separation and met system suitability; relative retention times (RRTs) of the characteristic peaks were within ±10% of specified values. Laboratories may choose among these.
2.2.2 Mobile phase
Methanol–0.1% formic acid yielded poor separation for most peaks except echinacoside; therefore, acetonitrile–0.1% formic acid was adopted and optimized to the gradient in Section 1.3, affording optimal resolution, plate numbers, and a stable baseline. As no further peaks eluted after 30 min, re‑equilibration was started at the initial composition.
2.3 Precision, repeatability, and stability
Using batch PFKL1, the test solution prepared per Section 1.2.2 was injected six times under Section 1.3 conditions. RSDs of RRTs and relative peak areas for the nine characteristic peaks were all <1%, indicating good instrumental precision. Six independently prepared test solutions of PFKL1 yielded RSDs <1% for both RRTs and relative peak areas, demonstrating repeatability. Stability testing at 0, 3, 9, 14, 21, and 28 h showed RSDs <1% for RRTs and relative peak areas, indicating the test solution was stable within 0–28 h.
2.4 Establishment of the characteristic chromatogram and technical parameters
2.4.1 Reference characteristic chromatogram
Ten batches of C. deserticola formula granules consistently displayed nine characteristic peaks that corresponded in retention time to nine peaks in the reference herb solution (Figures 1–2). Five peaks were assigned by standards as echinacoside, cistanoside A, tubuloside A, verbascoside (acteoside), and isoverbascoside. The echinacoside peak contributed a large and stable proportion of total peak area and was selected as the reference substance. The chromatographic behavior across ten batches was essentially consistent.
A.Blanksolution;B.Echinacoside;C.Testsolution;D.Cistanchedeserticola;E.Referencecharacteristicchromatogram.
Cistanche Tubulosa Specs List
2.4.2 Peak identification
Test solutions (Section 1.2.2) were analyzed by LC–MS in both positive and negative ESI modes; the positive mode provided richer peak information and was used (Figure 3). Nine compounds were identified in total; five were confirmed by exact mass, retention time, and MS/MS fragments using standards (Table 1). Given that PhGs are the main actives in C. deserticola and that the formula granules are prepared by aqueous extraction, LC/Q‑TOF‑MS confirmed PhGs as the main constituents. Accordingly, nine characteristic peaks were catalogued, and five peaks with relatively strong responses were annotated in the reference chromatogram.
Fig.2 HPLCreferencecharacteristicchromatogram 3(S):Echinacoside;4:CistanosideA;5:TubulosideA; 6:Acteoside;7:Isoacteoside.
Fig.3 Extractionioncurrentchromatogramoftestsolution 3(S):Echinacoside;4:CistanosideA;5:TubulosideA;6:Acteoside;7:Isoacteoside.
2.4.3 Specified relative retention times
Based on the ten batches, mean RRTs (relative to the S peak, i.e., echinacoside) were: 0.77 (Peak 1), 0.86 (Peak 2), 1.00 (Peak 3, S), 1.17 (Peak 4), 1.27 (Peak 5), 1.32 (Peak 6), 1.44 (Peak 7), 1.67 (Peak 8), and 1.81 (Peak 9). Each peak's RRT should fall within ±10% of its specified value.
Table 1. Chromatographic Peak Mass Spectrometry Parameters Related to Cistanche deserticola Formula Granules
| Peak | Name | Molecular Formula | m/z | Retention Time (min) |
|---|---|---|---|---|
| 1 | Unknown | C₅₁H₃₆O₁₁ | 824.2258 | 6.123 |
| 2 | Unknown | C₃₆H₃₀O₈ | 590.1941 | 6.876 |
| 3 | Echinacoside | C₃₅H₄₆O₂₀ | 809.2467 | 13.646 |
| 4 | Cistanoside A | C₃₆H₄₈O₂₀ | 823.2642 | 16.285 |
| 5 | Tubuloside A | C₃₇H₄₈O₂₁ | 851.2570 | 17.947 |
| 6 | Acteoside | C₂₉H₃₆O₁₅ | 647.1931 | 18.464 |
| 7 | Isoacteoside | C₂₉H₃₆O₁₅ | 647.1956 | 20.083 |
| 8 | Unknown | C₃₁H₃₈O₁₆ | 689.2044 | 23.446 |
| 9 | Unknown | C₃₁H₃₈O₁₆ | 689.2041 | 25.883 |
2.5 Similarity evaluation
Chromatograms from the ten batches were imported into the "Similarity Evaluation System for Chromatographic Fingerprint of TCM (2012A)." Using sample S1 as the reference, the median method, a 0.5‑min time window, multi‑point calibration, and data matching generated a reference fingerprint (Figure 4) with nine common peaks. Similarity values for all ten batches were ≥0.981 (0.981–0.999), indicating high inter‑batch similarity.
2.6 Transfer of quantity and quality from decoction pieces to standard decoction and formula granules
Peak matching showed that C. deserticola decoction pieces, standard decoction, and formula granules shared nine characteristic peaks, including the five assigned peaks (echinacoside, cistanoside A, tubuloside A, verbascoside, and isoverbascoside) (Figure 5). The characteristic profiles were highly correlated, and transfer rules from decoction pieces → standard decoction → formula granules were clear. From decoction pieces to standard decoction, the area of Peak 6 (verbascoside) decreased markedly, consistent with the heat‑lability of verbascoside.
Fig.4 Overlay of HPLC Characteristic Chromatogram of Cistanche deserticola Formulagranules
2.7 Comparison of characteristic chromatograms between C. deserticola and C. tubulosa formula granules
Both C. deserticola and C. tubulosa contain echinacoside, cistanoside A, verbascoside, isoverbascoside, and tubuloside A. However, the two species differ significantly in echinacoside content, and cistanoside A is present only at trace levels in C. tubulosa. Therefore, echinacoside and cistanoside A can serve as discriminatory markers for differentiating the botanical origin (C. deserticola vs. C. tubulosa).
Fig.5 Comparison ofcharacteristicchromatogramsof Cistanchedeserticoladecoctionpieces(A),standarddecoction(B)andformulagranules(C) 3(S):Echinacoside;4:CistanosideA;5:TubulosideA;6:Acteoside;7:Isoacteoside.
Conclusion (modified as requested: which species has "better" content)
Within the framework of the established HPLC–MS characteristic profile for Cistanche deserticola formula granules, and considering the inter‑species comparison, C. deserticola exhibits a richer and more balanced phenylethanoid glycoside profile: cistanoside A is readily detected in C. deserticola but is only trace in C. tubulosa, while echinacoside shows marked inter‑species differences. Therefore, for formulations aiming at higher aggregate levels of key PhGs and stronger overall cistanche benefits, Cistanche deserticola is recommended as the better species in terms of content. If a product specifically targets maximal echinacoside, selected batches of C. tubulosa will be considered depending on supply and lot testing. In practice, "best cistanche for sale" should be determined by batch‑specific quantitative testing of echinacoside, verbascoside (acteoside), and cistanoside A under the validated method described herein.
References
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Notes
Names of compounds: echinacoside; cistanoside A; tubuloside A; verbascoside (acteoside); isoverbascoside.
Apparent typographical slips referring to "nutmeg" in the source were rendered as "Cistanche" in translation based on context.
