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Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.

A tissue-smashing based ultra-rapid extraction coupled with ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed to determine 10 major triterpenoid saponins from Pulsatilla herbs. Compound 4 was characterized as betulinic acid glycoside 3-O-α-arabinopyranosyl-28-O-β-glucopyranosyl-23-hydroxy with HR-ESI-MS, 1H-NMR and 13C-NMR experiment. The MS spectra result showed that the ionization of compound 4 was more efficient in the positive mode. Meanwhile, the ions at m/z 789.6 and m/z 627.5 were selected as precursor and product ion for the determination, respectively. The chromatographic separation was carried out on a Phenomenex Kinetex C18 column using a gradient mobile phase system composed of 0.1% formic acid both in methanol and water at a flow rate of 0.4 mL/min. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive and negative mode. The total run time was 6 min. The calibration curves possessed good linearity with all coefficients higher than 0.9987. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 97.6% to 103.4% with RSD

C-5-substituted and C-8-substituted furanocoumarin isomers, two important kinds of furanocoumarin, are widely documented as the main active constituents in Angelicae dahuricae radix. Due to the similar polarity and mass fragmentation pathways of such isomers, it is difficult to distinguish them using mass spectrometric methods. To address this issue, we developed a strategy employing combined full scan and product ion scan modes on an ultra high performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) platform to differentiate four pairs of furanocoumarin isomer, viz. xanthotoxin and bergapten, imperatorin and isoimperatorin, psoralen and isopsoralen, and impinellin and isoimpinellin. A novel method using the ratio of relative abundance (RRA) of characteristic fragment ions was established to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, using the formula R = M/N, where M and N represent the ratios of relative abundance of characteristic fragment ions of a pair of furanocoumarin isomers. For R value greater than 1, compound M is substituted at C-5, whereas for R value less than 1, compound M is substituted at C-8. This method with good repeatability was applied to identify five pairs of isomeric furanocoumarins in Angelicae dahuricae radix. This is the first method to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, and can be used in complex matrix.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

Carbon black nanoparticles (CBNPs) are one of atmospheric particles components and have been closely related with a series of lung diseases. It can reach the depths of the respiratory tract or even alveolar more easily than those micro-particles. Although some of its toxicities have been confirmed in animals or human bodies, the subchronic toxicity mechanism of CBNPs has been uncertain so far. Therefore, it is very necessary to establish a novel method and clarify the mechanism of subchronic toxicity caused by concentration adjustments of small molecule metabolites in vivo. In animal experiments, CB exposure, recovery and control group were set up. The concentration of CBNPs in chamber was 30.06 ± 4.42 mg/m3. We developed a UHPLC-Q-TOF-MS/MS-based non-targeted metabolomic analysis strategy to analyze serum samples of rats. Then, differential metabolites in serum were found by multivariate data analysis and 39 potential biomarkers were identified. It was showed that main metabolic pathways associated with CBNPs exposure were hormones metabolism, amino acid metabolism, nucleotide metabolism and lipid metabolism. It is worth noting that long-term exposure to CBNPs had the greatest impact on steroid hormones biosynthesis so that the risk of infertility could increase. The results provided a new mechanistic insight into the metabolic alterations owing to CBNPs induced subchronic toxicity.

(+)-usnic acid (UA) is an active natural phenolic acid ingredient originating from Chinese traditional Tibetan herb. Usnea acid is expected to become a new agent for anticancer and remarkable antitumor. To reveal its metabolic profile, metabolites identification of UA in vivo was studied using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) in this present study. The chromatographic separation was performed on a C18 column with a mobile phase consisted of methanol and water with a flow rate of 0.4 ml/min. The mass spectral analysis conducted in a negative electrospray ionization mode combined with information-dependent acquirement technology (IDA) was used to trace all the potential UA metabolites. Several sensitive and specific multiple data-mining techniques especially key product ions (KPIs) filter were applied to hunt and identify metabolites rapidly. As a result, a total of 36 metabolites were detected after oral administration of UA, including 33, 8 and 16 in rat urine, plasma and bile, respectively. These results showed that the probable metabolite pathways of UA were oxidation, reduction, dihydroxylation, glycine conjugation, glucuronide conjugation, N-acetylcysteine conjugation and methylation. It is the first time to elucidate the profile of UA in vivo. These results not only provided the basis of UA pharmacological properties, but also gave the guidance in clinical medication. Moreover, the analysis strategy and methodology proposed in this paper could be widely used in characterization of other phenolic acids metabolites.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of nine coumarins including aesculin, aesculetin, fraxin, fraxetin, scopoletin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarin and umbelliferone in rat plasma using nodakenin as the internal standard (IS). The plasma samples were pretreated by a one-step direct protein precipitation with methanol. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and water (containing 0.05% acetic acid). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. This method was fully validated in terms of the sensitivity, specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analyte under various conditions, and the results satisfied the requirements of biological sample measurement. The validated method was successfully applied to pharmacokinetic study of the nine coumarins in rat plasma after oral administration of Fraxini Cortex aqueous extract, among which the pharmacokinetics of four coumarins including fraxetin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxy coumarin were studied for the first time.

The detection of drug metabolites, particularly for minor metabolites, continues to be a challenge owing to the complexity of biological samples. Imperatorin is an active natural furocoumarin ingredient originating from many traditional Chinese herbal medicines. In the present study, the metabolites of imperatorin after oral administration were qualitatively investigated, and possible metabolic pathways of it were subsequently proposed. Bile samples were collected after oral administration and pretreated by the application of Waters Ostro. The QTOF-MS/MS data was acquired using ultra high performance liquid chromatography coupled to quadrupole time flight spectrometry (UPLC-QTOF-MS). Based on this analytical strategy, 32 metabolites (23 phase I and 9 phase II metabolites) were identified in rat bile. The results demonstrated that C5H8 could be easily eliminated from imperatorin forming the metabolite M1. It also indicated that imperatorin and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. This is the first study of imperatorin metabolism in bile samples. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of other linear-type furocoumarins.

Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rat plasma using sulfamethoxazole as an internal standard (IS). Separation was conducted out on an Agilent Eclipse XDB C18 column with liner gradient elution using acetonitrile (A) and 0.1% aqueous acetic acid (B). A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting three scanning periods. All analytes exhibited good linearity within the concentration range (r>0.9973). The lower limits of quantitation (LLOQ) of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin were 2.64, 4.32, 2.32 and 1.56ng/mL, respectively. Intra-day and inter-day precisions of the investigated components exhibited an RSD within 8.3%, and the accuracy (RE) ranged from -8.6% to 6.0% at all quality control levels. The developed method was successfully applied to a pharmacokinetic study of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rats after oral administration of a Weifuchun tablet.

Introduction Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. Objective To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. Methodology In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. Results As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. Conclusion This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.

Introduction The Kang-nao-shuai (KNS) capsule is a combined herbal prescription used in the treatment of insomnia, amnesia, neurasthenia, age-related dementia and brain injuries. Multiple constituents are considered to be responsible for the therapeutic effects of this herbal prescription. However, the quality control of the multicomponents is limited. Objective To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for the analysis of 40 constituents in KNS capsules. Methodology The optimal chromatographic conditions were achieved on an Agilent C18-column with a gradient elution that consisted of methanol and 0.1% formic acid in water. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer in positive and negative mode respectively using multiple-reaction monitoring. Results A total of 40 constituents including organic acid, flavonoid, quinone, terpene, alkaloid and saponin were quantified, most of the 40 components were determined for the first time in the KNS capsule. A quantitative HPLC–ESI–MS/MS method allowing the quantification of 40 marker compounds was optimised and validated for linearity, precision, accuracy, stability, specificity and limits of detection and quantification. The method was successfully applied to analyse 10 batches of KNS capsule. Conclusion The established method is simple and can be used as a tool for quality evaluation and control of this natural product. Copyright © 2011 John Wiley & Sons, Ltd.

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring–information dependent acquisition–enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.

A sensitive, specific, and rapid liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for analysis of lasiodonin, oridonin, ponicidin, and rabdoternin A in rat plasma using sulfamethoxazole as an internal standard (IS). The plasma samples were pretreated and extracted by liquid–liquid extraction. Chromatographic separation was performed on a C 18 column with linear gradient elution using water and methanol, both of which were acidified with 0.1% aqueous formic acid, at a flow rate of 0.8 ml/min. Detection was accomplished by scanning with multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion pairs ( m / z ) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for all analytes in the rat plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon rubescens extract to rats.

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi-components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C18 column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow-rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.

A novel method, HPLC-MS/MS was developed to qualitatively identify and quantitatively determine the 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine. The separation was performed on a C(18) column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7 mL/min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, LOD and LOQ). The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 28 chemical compositions in 21 batches of natural and cultured I. rubescens samples from different sources which had great variation on the contents. The results demonstrated that the method was useful for standardization and differentiation of large numbers of similar samples.

OBJECTIVE To establish a HPLC-DAD method for determining concentrations of betulic acid and phenol red in intestinal circulation liquid, and probe into the absorption kinetic characteristics of betulic acid at different intestine segments in rats and the effect of different drug concentrations on absorption. METHOD The rat intestinal absorption model was established to detect the impact of absorption site, drug concentration and pH value on drug absorption. RESULT Within the range from 75-125 mg x L(-1), the absorption rate and the quality concentration of betulic acid had a linear relation, with Ka value keeping unchanged. The absorption rate for each intestinal segment showed no remarkable difference, with Ka values in duodenum, jejunum, ileum and colon being (0.151 +/- 0.0049), (0.159 +/- 0.0056), (0.156 +/- 6.0083), (0.149 +/- 0.0041) h(-1), respectively. CONCLUSION Betulic acid is proved to be well absorbed in intestines marked by no specific absorption site in the intestine. The absorption mechanism of the drug conforms to passive transport mechanism and first-order kinetics. The bioavailability of betulic acid preparation can be increased by enhancing the dissolution rate and the solubility.

Dipfluzine hydrochloride (Dip), a novel diphenylpiperazine calcium channel blocker, has revealed the characteristics of a promising candidate for the treatment of cerebral vascular diseases in preclinical studies. Our research identified and quantified Dip and its 4 metabolites (M1, M2, M4 and M5) in rat liver microsomes by liquid chromatography tandem mass spectrometry. The results showed that Dip was firstly metabolized to M1 and M5 by 1- and 4-dealkylation from a piperazine nitrogen, and then the latter was subsequently metabolized to M2 and M4. The concentrations of Dip, M1, M2 and M5 were 557.3 ± 26.3, 854.3 ± 46.0, 2796.7± 126.9, 2473.3 ± 82.6 and 4.0 ± 0.4, 2.4 ± 0.1, 318.2 ± 8.7 and 27.4 ± 1.5 ng/ml in male and female rats, respectively. M4 (404.2 ± 22.2 ng/ml) was detected only in males not in females, suggesting that there is gender difference in the metabolism of Dip.

A simple and sensitive LC-MS/MS method has been developed and validated for the identification and quantification of epinodosin, epinodosinol, nodosin, oridonin, lasiokariurinol, lasiokaurin and rabdoternin A in rat plasma using sulfamethoxazole as the internal standard. The plasma sample pre-treatment consisted of a liquid-liquid extraction. Chromatographic separation was achieved on a C18 column with linear gradient elution using water and methanol, which were both acidified with 0.1% formic acid, at a flow rate of 0.7 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting five scanning periods. The method presented here utilizes a novel determination strategy, enabling the application of positive and negative ESI-MS in a single run. The optimized mass transition ion-pairs (m/z) for quantitation were 361.2/287.1 for epinodosin, 382.3/347.3 for epinodosinol, 363.3/281.2 for nodosin, 365.3/347.3 for oridonin, 407.3/329.1 for lasiokariurinol, 405.2/59.0 for lasiokaurin, 363.2/283.1 for rabdoternin A and 254.1/156.0 for IS. The total run time was 20.50 min (including 5 min equilibration time) between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect and several validation results demonstrate that this method is sensitive, specific and reliable. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon serra extract to rats.

A high-performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method was developed to characterize and quantify 19 diterpenoid compounds in Isodon amethystoides simultaneously. By employing a Diamonsil C18 column, 19 constituents were separated within 15 min using a gradient elution consisted of methanol containing 0.1% formic acid and 0.1% aqueous formic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode in a single run and quantified by a multiple-reaction monitor (MRM). All standard calibration curves showed good linearity (r2 > 0.99) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.06–3.25% and 1.56–3.84%. The recovery studies for the quantified compounds were between 95.82 and 108.3% with RSD values less than 1.86%. The results indicated that the method is simple, rapid, specific and reliable. This method was successfully applied for identification and quantification of 19 diterpenoids in 11 batches of I. amethystoides. The results showed that the contents of diterpenoids in I. amethystoides from different sources were widely varied.