Fragmentation study of iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae by rapid resolution liquid chromatography with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid resolution liquid chromatography (RRLC) coupled with diode array detection (DAD) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) method was applied to the mass spectral study of a series of naturally occurring iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae, which provides higher speed and increased sensitivity without loss of resolution. With dynamic adjustment as the key role of the fragmentor voltage and confirmed with authentic standards, valuable structural information regarding the nature of both the glycoside skeletons was thus obtained. Most compositions were found to possess organic acid moiety such as cinnamoyl, caffeoyl and ferulyol. Besides extensive fragmentation of the carbohydrate moiety, losses of the hydroxyl and glucose residue units showed in the spectra, permitting the exploration of the skeleton and the identity of substituents in the molecule. Ten major iridoid glycosides and 10 phenylpropanoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in Radix Scrophulariae obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance spectrometry and liquid chromatography with nuclear magnetic resonance and mass spectrometric detection

This paper describes the first application of NMR spectroscopy and LC-NMR/MS to the direct analysis of the aromatic composition of beer, grape juice and a wine phenolic extract. NMR spectroscopy provides non-invasive information on the overall aromatic profile and enables the identification of some compounds. However, a more comprehensive assignment is hindered by the low peak intensity and strong signal overlap in the low-field spectral region, as well as by the inherent lack of scalar coupling information for many aromatic compounds present. LC-NMR/MS can overcome these problems and is shown to aid significantly in the identification of aromatic compounds composing all samples analyzed. Some examples are the identification of several cinnamic acids (e.g. p-coumaric, trans-coutaric and trans-caftaric) in grape juice, the identification of 2-phenylethanol, tyrosol and tryptophol in beer and the detection of phenolics such as catechin, epicatechin, trans-resveratrol, tyrosol and caffeic acid in the wine extract.     

Fallaxosides B1 and D3, triterpene glycosides with novel skeleton types of aglycones from the sea cucumber Cucumaria fallax

Two unprecedented triterpene glycosides, fallaxosides B₁ and D₃ with aglycones of new skeletal types were isolated from the sea cucumber Cucumaria fallax (Cucumariidae, Dendrochirotida). The first of these aglycones is formed as result of intramolecular aldol condensation of a precursor having 1,6-diketone functionality, while the second one by pinacol-pinacolone-like rearrangement of another precursor, probably containing 7,8,9-trihydroxy fragment (or its 8,9-epoxy-7-hydroxy analog). The structures of these compounds were established by analysis of 1D, 2D NMR, ESI MS and CD data with using the quantum-chemical calculations.     

Combination of a new sample preparation strategy with an accelerated high-performance liquid chromatography assay with photodiode array and mass spectrometric detection for the determination of destruxins from Metarhizium anisopliae culture broth

A method is presented allowing the qualitative and quantitative analysis of destruxins (dtxs) in fungal culture broth. Sample preparation was carried out by ultrafiltration over a commercially available acetylated cellulose (CTA) membrane with a Mr 10000 cut-off. The developed high-performance liquid chromatography assay with diode array detection (HPLC-DAD) cuts down the analysis time by 50% compared to most of the currently applied methods (retention times: dtx A = 8.3 min, dtx B = 8.9 min, dtx E= 7.5 min) and enables dtx detection down to sub-ppm range (limits of detection: dtx A = 0.19 mg/l, dtx B = 0.41 mg/l, dtx E = 0.10 mg/l). Stability of dtx E in filtrated culture broth was found to be much lower than anticipated (half-life time = 64.5 +/- 1.7 h). Thus, the detoxification of this metabolite is an abiotic process. Coupling of the HPLC-DAD system to an ion trap mass spectrometer with an electrospray ionization source operating in the positive mode allowed identification of most dtxs encountered by utilizing multiple stage MS-MS experiments and retention time rules.     

A novelty strategy for the fast analysis of sulfonamide antibiotics in fish tissue using magnetic separation with high‐performance liquid chromatography–tandem mass spectrometry

A simple, fast and low‐cost extraction method with high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) determination was developed on sulfonamide antibiotics (SAs) in fish tissue. Magnetic separation was first introduced into the rapid sample preparation procedure combined with acetonitrile extraction for the analysis of SAs. Partitioning was rapidly achieved between acetonitrile solution and solid matrix by applying an external magnetic field. Acetonitrile solution was collected and concentrated under a nitrogen stream. The residue was redissolved with 1‰ formic acid aqueous solution and defatted with n‐hexane before analysis. The recoveries of SAs were in the range of 74.87–104.74%, with relative standard deviations <13%. The limits of quantification and the limits of detection for SAs ranged from 5.0 to 25.0 μg ^kg?1 and from 2.5 to 10.0 μg ^kg?1, respectively. The presented extraction method proved to be a rapid method which only took 20 min for one sample preparation procedure. Copyright © 2016 John Wiley & Sons, Ltd.     

Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D₄ and 5- methyl tetrahydrofolic acid ¹³C₅, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.     

The application of high performance liquid chromatography, coupled to nuclear magnetic resonance spectroscopy and mass spectrometry (HPLC-NMR-MS), to the characterisation of ibuprofen metabolites from human urine

Summary The use of HPLC-NMR-MS for the detection and identification of the metabolites of ibuprofen present in a solid phase extract of human urine is described. Gradient reversed-phase HPLC was used to separate the components present in the extract, which were then characterised by a combination of stopped-flow¹H NMR and on line electrospray-MS. This approach led to the rapid identification of the known phase 1 human metabolites of ibuprofen, including hydroxy- and carboxy- metabolites, together with their respective glucuronide conjugates. In addition a probable artefact resulting from the dehydration of one of the side chainhydroxylated glucuronides was also identified.     

Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant,Tripterygium wilfordii,by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

In this study, a reliable method for analysis and identification of eight terpenoids in tissue cultures of Tripterygium wilfordii has been established using high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS). Our study indicated that sterile seedlings, callus cultures and cell‐suspension cultures can rapidly increase the amount of biological materials. HPLC‐ESI‐MS was used to identify terpenoids from the extracts of these tissue cultures. Triptolide, triptophenolide, celastrol and wilforlide A were unambiguously determined by comparing the retention times, UV spectral data, and mass fragmentation behaviors with those of the reference compounds. Another four compounds were tentatively identified as triptonoterpenol, triptonoterpene, 22β‐hydroxy‐3‐oxoolean‐12‐en‐29‐oic acid and wilforlide B, based on their UV and mass spectrometry spectra. The quantitative analysis showed that all three materials contain triptolide, triptophenolide, celastrol, wilforlide A, and the contents of the four compounds in the cell‐suspension cultures were 53.1, 240, 129 and 964 µg/g, respectively, which were at least 2.0‐fold higher than these in the sterile seedlings and callus cultures. Considering the known pharmacological activity of triptolide and celastrol, we recommend the cell‐suspension cultures as biological materials for future studies, such as clinical and toxicological studies. The developed method was validated by the evaluation of its precision, linearity, detection limits and recovery, and it was successfully used to identify and quantify the terpenoids in the tissue cultures. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacokinetic and bioavailability study of 5-hydroxy-4-methoxycanthin-6-one,a typical canthinone alkaloid,in rats using ultra-high performance liquid chromatography/electrospray ionization tandem mass spectrometry

5-methoxycanthin-6-one, a major canthinone alkaloid isolated from Picrasma quassioides, exhibited significant pharmacological activities. In this study, a rapid and sensitive LC–MS/MS method was established and validated for the determination of 5-hydroxy-4-methoxycanthin-6-one in rat plasma. Small quantities (20 μL) of plasma sample were used for sample preparation. 5-Hydroxy-4-methoxycanthin-6-one and an internal standard (IS, caffeine) were separated using an ACQUITY HSS T3 column (50 × 2.1 mm, 1.7 μm; Waters, Milford, MA, USA). The mobile phase was composed of 0.1% formic acid in water and acetonitrile. Precursor-to-product ion transitions were m/z 267.0 → 168.2 and m/z 195.0 → 138.1 for quantitative monitoring of 5-hydroxy-4-methoxycanthin-6-one and IS, respectively. The assay was linear over the concentration range of 0.5–500 ng/mL (r > 0.99) with the lower limit of quantification 0.5 ng/mL. Other parameters, including intra- and inter-day precision and accuracy, carryover, stability, extraction recovery, matrix effect, and dilution effect, were within acceptable limits. The validated method was successfully applied to pharmacokinetic study in rats after intravenous (5 mg/kg) and oral (10, 25, 50 mg/kg) administration of 5-hydroxy-4-methoxycanthin-6-one. The result indicated that 5-hydroxy-4-methoxycanthin-6-one was quickly absorbed into the blood and reached the highest concentration at ~33.0–42.0 min, with moderate elimination half-life (0.85–2.11 h) and low bioavailability (16.62–24.42%) after oral administration. The study provided valuable information that can be used as a reference for studying other canthinone alkaloids.     

Characterization of degradation products of poly[(3,3,3-trifluoropropyl)methylsiloxane] by nuclear magnetic resonance spectroscopy, mass spectrometry and gas chromatography

Poly[(3,3,3-trifluoropropyl)methylsiloxane] (PTFPMS) was treated with the solvents acetone, ethyl acetate and methanol and its degradation products were analyzed with multiple techniques. ¹H, ²⁹Si and ¹⁹F nuclear magnetic resonance (NMR) spectroscopy were useful for the characterization of the intact polymers and for the determination of cyclosiloxanes. Cyclosiloxanes with a ring size of up to 23 were quantified by gas chromatography. The only degradation products found were TFPmethyl-cyclosiloxanes. 1,3,5,7-Tetrakis(TFPmethyl)-cyclotetrasiloxane was predominant, and (TFPmethyl)cyclotri-, penta- and hexasiloxane could be detected at lower concentrations. The identity of cyclic degradation products with a ring size of up to 6 was unambiguously confirmed by direct infusion mass spectrometry. The TFPmethyl-cyclosiloxanes were successfully ionized by electrospray ionization in the negative mode. None of the techniques applied gave hints to other degradation products such as short linear oligomers. Almost complete degradation of PTFPMS occurred in acetone and methanol, while degradation is distinctly reduced in ethyl acetate.     

Analytical investigation of secondary metabolites extracted from Camellia sinensis L. leaves using a HPLC‐DAD‐ESI/MS data fusion strategy and chemometric methods

Considerable attention has been paid to the study of green tea leaves because of their high consume and beneficial effects on human health. In this work, an appropriate strategy is proposed to investigate and resolve the major metabolites extracted from Camellia sinensis tea leaves. Statistical design mixtures of ethanol, ethyl acetate, dichloromethane, and chloroform were used to study the effects of different solvents and their mixtures on the extraction of the secondary metabolites of C. sinensis tea leaves from two different harvest seasons. Extracted samples were analyzed by high performance liquid chromatography‐diode array detection‐electrospray ionization mass spectrometer allowing the resolution of a large amount of tea metabolites with high relative abundances, especially when their extraction was performed in pure ethanol and with solvent mixtures with ethanol. Resolution of the more relevant metabolites was achieved by the simultaneous analysis of the fused diode array detection and mass spectrometer detectors data from the same samples using the multivariate curve resolution‐alternating least squares chemometric method. Peak areas finally resolved were further analyzed by orthogonal signal correction and partial least squares‐discrimination analysis to discriminate among C. sinensis tea samples. Using the Variable Importance in Projection variable selection method, epigallocatechin and caffeine were finally selected as the two more important chemical constituents of tea leaves that were discriminating more between the tea samples from two different harvest seasons. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of chemicals and their metabolites from PHY906, a Chinese medicine formulation,in the plasma of a patient treated with irinotecan and PHY906 using liquid chromatography/tandem mass spectrometry (LC/MS/MS)

Traditional Chinese Medicine (TCM) is increasingly being used in combination with Western medicine. In general, TCM is comprised of multiple components in sharp contrast to Western medicine, where a single active chemical is used. Presently, there are no well-established standards for most of the chemical compounds of TCM and their respective metabolites. Moreover, there are no formal analytical methods for the identification of these chemicals, especially in trace amounts. The ability to measure the pharmacokinetic behaviors of chemicals and their metabolites from these herbal formulations are critical in understanding of the action of TCM. This paper describes the use of LC/MS/MS along with enzyme treatments and n-octanol/water partition coefficient, to investigate the chemical components of PHY906 and their metabolites in the plasma of a patient with metastatic colorectal cancer (mCRC) treated with irinotecan and PHY906. The chemicals from an aqueous extract of PHY906 and the plasma from a patient was prepared and separated on an Agilent ZORBAX-SB C₁₈ column, and eluted with acetonitrile/0.05% (v/v) formic acid. From the PHY906 aqueous extract, a total of 57 compounds and 27 metabolites were identified and tentatively assigned structures based on their identified mass spectrometry, enzyme digestion and n-octanol/water partition coefficient. In contrast, analysis of patient plasma identified only 33 chemicals and new metabolites. These findings demonstrated that LC/MS/MS was and effective and reliable method for studying the parent chemicals of the Chinese herbal medicine PHY906 and their metabolites in a patient with metastatic colorectal cancer.     

Screening, identification and quantification of glucosinolates in black radish (Raphanus sativus L. niger) based dietary supplements using liquid chromatography coupled with a photodiode array and liquid chromatography-mass spectrometry

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48 mg/g), glucosisaustricin (0.37-0.91 mg/g), glucoraphenin (0.84-1.27 mg/g), glucoputrajivin (0.14-0.28 mg/g), glucosisymbrin (0.70-0.99 mg/g) and gluconasturtiin (0.06-0.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.     

Sensitive quantification of coixol,a potent insulin secretagogue,in Scoparia dulcis extract using high‐performance liquid chromatography combined with tandem mass spectrometry and UV detection

Diabetes is a major global health problem which requires new studies for its prevention and control. Scoparia dulcis , a herbal product, is widely used for treatment of diabetes. Recent studies demonstrate coixol as a potent and nontoxic insulin secretagog from S. dulcis . This study focuses on developing two quantitative methods of coixol in S. dulcis methanol‐based extracts. Quantification of coixol was performed using high‐performance liquid chromatography–tandem mass spectrometry (method 1) and high‐performance liquid chromatography–ultraviolet detection (method 2) with limits of detection of 0.26 and 11.6 pg/μL, respectively, and limits of quantification of 0.78 and 35.5 pg/μL, respectively. S. dulcis is rich in coixol content with values of 255.5 ± 2.1 mg/kg (method 1) and 220.4 ± 2.9 mg/kg (method 2). Excellent linearity with determination coefficients >0.999 was achieved for calibration curves from 10 to 7500 ng/mL (method 1) and from 175 to 7500 ng/mL (method 2). Good accuracy (bias < −8.6%) and precision (RSD < 8.5%) were obtained for both methods. Thus, they can be employed to analyze coixol in plant extracts and herbal formulations.     

Liquid chromatography mass spectrometry for the determination of salvianolic acid B,a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study

A sensitive and specific liquid chromatographic–electrospray ionization mass spectrometric method was developed for quantification of salvianolic acid B in rat plasma with resveratrol as the internal standard. The analytes were separated on a reversed‐phase column with acetonitrile (40%) and water (60%) containing 0.75% formic acid as mobile phase at a flow rate of 1 mL/min. Liquid–liquid extraction was adopted for the sample preparation, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode. The method was validated over the concentration range 0.1–40 µg/mL using 0.1 mL of plasma with coefficients of correlation >0.999. The intra‐ and inter‐day precisions of analysis were <10%, and accuracy ranged from 94 to 101%. This method was successfully applied to a pharmacokinetics of salvianolic acid B in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Rapid collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric analysis

The present research reports the use of a three-dimensional preparative gas chromatography (prep GC) system, equipped with three Deans-switch devices and 5%diphenyl/wax/mid-polarity ionic liquid stationary phases, for the isolation of volatile components from a complex natural source, namely wampee essential oil (derived from Clausena lansium Skeels leaves). Collection was performed by using a simple and effective lab-constructed trapping device.     

In vivo metabolism study of rhubarb decoction in rat using high-performance liquid chromatography with UV photodiode-array and mass-spectrometric detection: a strategy for systematic analysis of metabolites from traditional Chinese medicines in biological samples

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.     

On-line identification of 4″-isovalerylspiramycin I in the genetic engineered strain of S. spiramyceticus F21 by liquid chromatography with electrospray ionization tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC–DAD-UV–ESI-MSⁿ analysis played a crucial role, and stop-flow LC–¹H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development.     

Qualitative and quantitative analysis of cinobufacini injection using rapid separation liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and HPLC‐photodiode array detection,a feasible strategy for the quality control of Chinese medicine injections

Cinobufacini injection, prepared from the skin of Bufo bufo gargarizans Cantor, has presented its significant effects on the treatment of hepatitis and various cancers in the clinic. However, as an unclear complex chemical system, the optimization of its quality control markers has been a long‐term challenge. In present study, a feasible strategy integrated markers screening, determination, and statistical analysis was efficiently proposed, especially for the undefined Chinese medicine injections. First, rapid separation LC‐quadrupole‐TOF‐MS method was applied in the identification of 19 major compounds in the cinobufacini injection for the first time. Further, nine high‐level contents active compounds were selected as quality control markers for the quantification analysis. An acceptable and validated determination method was established in 17 batches of cinobufacini injection by HPLC‐photodiode array detection method, including linear regression relationship (r², 0.9996–1), precisions (RSD, 0.02–1.35%), repeatability (RSD, 0.05–1.97%), stability (RSD, 0.1–3.85%), and recovery (95.88–104.89%). Each analyte was detected at its maximum ultraviolet spectra wavelength. Finally, based on the quantification results, principal component analysis was performed on the quality assessment of cinobufacini injections. This three‐step strategy provides a newly feasible solution for the quality control of Chinese medicine injections.