A novel strategy to rapidly explore potential chemical markers for the discrimination between raw and processed Radix Rehmanniae by UHPLC–TOFMS with multivariate statistical analysis

In traditional Chinese medicine, raw and processed herbs are used to treat different diseases. Suitable chemical markers are crucial for the discrimination between raw and processed herbs. In this study, a novel strategy using UHPLC–TOFMS coupled with multivariate statistical analysis to rapidly explore potential chemical markers was proposed and validated. Using Radix Rehmanniae as a model herb, batches of raw and processed samples were determined by UHPLC–TOFMS. The datasets of t_R–m/z pair, ion intensity and sample code were subjected to principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between raw and processed samples. Once a clear cluster was found, extended statistics was performed to generate S-plot, in which the variables (t_R–m/z pair) contributing most to the difference were clearly indicated as points at the two ends of “S”, and the components that correlate to these ions should be the processing-induced transformed components. These transformed components could be regarded as the potential chemical markers that can be used to distinguish between raw and processed herbs. The identity of the potential markers can be identified by comparing the mass/UV spectra and retention time with that of reference compounds and/or tentatively assigned by matching empirical molecular formula with that of the known compounds published. Using this proposed strategy, leonuride or its isomer and 5-(α-d-glucopyranosyl-(1-6)-α-d-glucopyranosyloxymethyl)-2-furancarboxaldehyde were rapidly explored as the most characteristic markers of raw and processed Radix Rehmanniae, respectively. This newly proposed strategy can not only be used to explore chemical markers but also to investigate the chemical transforming mechanisms underlying traditional herb processing.     

Metabolomic profiling of Saposhnikoviae Radix from Mongolia by LC–IT–TOF–MS/MS and multivariate statistical analysis

Journal of Natural Medicines - Saposhnikoviae Radix (SR) is a commonly used crude drug that is obtained from the root and rhizome of Saposhnikovia divaricata which is distributed throughout China,...     

Simultaneous analysis of bioactive metabolites from Ziziphus jujuba by HPLC–DAD–ELSD–MS/MS

A high-performance liquid chromatography (HPLC) with diode array detector (DAD), evaporative light scattering detector (ELSD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of nine representative metabolites of the alkaloid, flavonoid and triterpenoid classes from Ziziphus jujuba var. spinosa. The optimal chromatographic conditions were obtained on an ODS column and the mobile phase was composed of water and methanol with 0.1% formic acid using a gradient elution system. Using the developed methods, all of the validation parameters were successfully obtained. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of representative metabolites in commercial samples of Z. jujuba and Z. mauritiana from different markets in Korea, China and Myanmar. The analytical results showed that the contents of the nine analytes vary significantly with sources and species, thus demonstrating its potential for the detection of this plant.     

Identification of multiple constituents in the traditional Chinese medicine formula GuiZhiFuLing-Wan by HPLC–DAD–MS/MS

GuiZhiFuLing-Wan (GFW) has been used in China for centuries to improve blood stagnation. In this paper, a HPLC–DAD–MS/MS method was established for the efficient and rapid identification of the chemical constituents in extract of GuiZhiFuLing-Wan. Separation was performed on an Alltima C₁₈ analytical column by gradient elution with CH₃CN/H₂O–CH₃COOH as mobile phase at a flow rate 1.0 ml/min. 27 potentially bioactive compounds including monoterpene glycosides, galloyl glucoses, acetophenones, phenylallyl compounds and triterpenoids were identified or tentatively characterized by online ESI/MS/MS and the comparison with literature data and authentic compounds. After the identification, six different brands of GFW commercial products in various dosage forms were evaluated. The results demonstrated that capsule of GFW was superior to the other two dosage forms, honeyed pill and concentrated pill in administration. The points that should be paid more attention during the manufacturing process of GFW were also analyzed. The method can be the basis for the quality control of this commonly used herbal formula.     

Determination of mangiferin in rat plasma by liquid–liquid extraction with UPLC–MS/MS

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for the determination of mangiferin in rat plasma using diphenhydramine as the internal standard (IS). Liquid–liquid extraction (LLE) was used for sample preparation and the analysis was achieved with gradient elution on C₁₈ reversed phase column. The method was validated over the concentration range 0.02–5.0 μg/mL for oral administration and 0.4–100 μg/mL for intravenous administration. The intra-day and inter-day precision of mangiferin expressed as RSD < 15% and the accuracy (RE) did not exceed 15%. This validated method is a novel technique for sample preparation and quantitation, which was successfully applied to estimate the bioavailability of mangiferin.     

Simultaneous quantification of 19 ginsenosides in black ginseng developed from Panax ginseng by HPLC–ELSD

A high-performance liquid chromatographic method with evaporative light scattering detection (HPLC–ELSD) has been developed to identify and quantify 19 ginsenosides (Rg₁, Re, Rf, Rb₁, Rc, Rb₂, Rd, F₄, Rg₆, Rk₃, Rh₄, 20(S)-, 20(R)-Rg₃, 20(S)-, 20(R)-Rs₃, Rk₁, Rg₅, Rs₄, and Rs₅) in black ginseng (BG, Korean white ginseng that was subjected to nine cycles of steam treatment). Ultrasonication is employed for sample preparation, and the analysis is achieved on a Discovery C₁₈ column using gradient elution of CH₃CN–H₂O–CH₃COOH without buffer in 40 min. The method was validated by linearity (r² ≥ 0.9994), precision (92.0–107.5%), intra- and inter-day accuracy (R.S.D. < 3.21%), and limit of detection (LOD ≤ 93 ng). The quantification method was applied to analyze the composition of ginsenosides in Korean white, red, and black ginsengs. During the preparatory process of BG, ginsenosides transform into constituents of low polarity by hydrolysis, isomerization, and dehydration at C-20, and hydrolysis also occurs at C-3 or C-6. The validated HPLC method is expected to provide the basis for the quality assessment of ginseng products.     

Analysis of angiotensin II receptor antagonist and protein markers at microliter level plasma by LC–MS/MS

An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.     

Simultaneous Determination of Atorvastatin and Aspirin in Human Plasma by LC–MS/MS: Its Pharmacokinetic Application

A simple, rapid, and sensitive liquid chromatography tandem mass spectro-metric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin and aspirin in human plasma using a polarity switch. Proguanil and furosemide were used as the internal standards for the quantification of atorvastatin and aspirin, respectively. The analytes were extracted from human plasma by the liquid–liquid extraction technique using methyl tert-butyl ether. The reconstituted samples were chromatographed on a Zorbax XDB Phenyl column by using a mixture of 0.2% acetic acid buffer, methanol, and acetonitrile (20:16:64, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Prior to detection, atorvastatin and aspirin were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The ions were monitored at the positive m/z 559.2→440.0 transition for atorvastatin and the negative m/z 179.0→136.6 transition for aspirin. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.20–151 ng/mL for atorvastatin and 15.0–3000 ng/mL for aspirin. The method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 3.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Determination of fluoroquinolones in chicken feces – A new liquid–liquid extraction method combined with LC–MS/MS

The application of antibiotics including fluoroquinolones to farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects. To calculate environmental loads and for a proper risk assessment it is necessary to determine the antibiotic concentration in feces. Therefore, a new liquid–liquid extraction method combined with HPLC–MS/MS for the detection of marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin in chicken feces was developed. Recoveries ranged from 51.0% to 83.5%. LOQs were between 0.10 and 1.09 μg/kg. Feces of chickens treated with an enrofloxacin dosage of 10 mg/kg bodyweight revealed maximum enrofloxacin and ciprofloxacin concentrations of 61.3 and 18.8 mg/kg. Both antibiotics could be detected in feces up to two days after the last application in notable amounts (∼1 mg/kg). Thus, feces of recently medicated chickens should not be used as a fertilizer without any further processing.     

Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC–MS/MS with multiple reaction monitoring mode

Context: Fructus Corni, a traditional Chinese medicines, is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae). Gallic acid, 5-hydroxymethylfurfural, morroniside, sweroside, loganin, cornin, 7-O-methyl-morroniside and cornuside are the active constituents of Fructus Corni used in many traditional Chinese medicines. This paper describes a sensitive and specific assay for the determination of the eight bioactive compounds in crude and processed Fructus Corni extracts.

Materials and methods: In this paper, the eight components were determined by high performance liquid chromatography coupled with triple quadrupole mass spectrometry (MS/MS). Quantization was based on multiple reaction monitoring using the precursor production combination for determination of the eight analytes. The analysis was performed on an Agilent Zorbax Extend C₁₈ column (100?mm × 3.0?mm, 3.5 μm), and an electrospray ionization (ESI)-tandem interface in the positive and negative ion polarity mode was employed prior to mass spectrometric detection.

Results: With the optimized conditions, the eight bioactive compounds were detected properly within 10?min. The developed method showed good precision and reproducibility with the limits of detection ranged from 0.0042 to 12.7875?ng/mL and the average recoveries ranged from 97.08 to 103.7%.

Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for intrinsic quality control of crude and processed Fructus Corni.     

Simultaneous determination of five lignan constituents of Wuzhi capsule in rat plasma by LC–MS/MS: Application to pharmacokinetic study

A rapid sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC–MS/MS method. Liquid–liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100 mm × 2.1 mm i.d., 3.5 μm) by isocratic elution using a mobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at a flow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r² > 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule.     

Simultaneous quantification of 19 diterpenoids in Isodon amethystoides by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

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.     

Detection of clenbuterol residues in beef sausages and its enantiomeric analysis using UHPLC–MS/MS: A risk of unintentional doping in sport field

Clenbuterol (Clb) can be present in Mexico often but not all over the world in animal tissues and organs, therefore, potentially is derived from animal sources as well. The aims of this study were to develop and validate a method for detecting traces of clenbuterol in beef sausages. A calibration curve showed linearity in the range of 20–500 pg ml⁻¹. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 5 and 10 pg g⁻¹, respectively. The analyte recovery was from 95.70% to 100.40% with an intraday relative standard deviation (RSD%) of 0.99%–2.10% and an interday RSD% of 0.54%–2.34%, R² = 0.9998. The methodology developed was applied successfully in 15 samples of beef sausage, and 73.3% of the samples tested contained racemic clenbuterol in concentrations between 30 and 471 pg g⁻¹. The UHPLC–MS/MS method developed combines high sensitivity with good selectivity and short chromatographic run time. Additionally, the enantiomeric analysis of clenbuterol performed in beef sausages showed a 59% for R-(−)-Clb and 41% for S-(+)-Clb. The presence of clenbuterol in beef sausages could represent a risk of unintentional doping in sport field, because the clenbuterol is a banned substance included in the World Anti-Doping Agency's (WADA) list of prohibited substances.     

Decocting-induced chemical transformations and global quality of Du–Shen–Tang,the decoction of ginseng evaluated by UPLC–Q-TOF-MS/MS based chemical profiling approach

An UPLC–Q-TOF-MS/MS based chemical profiling method was developed to evaluate decocting-induced chemical transformations in Du–Shen–Tang, the decoction of the root of Panax ginseng. Under the optimized UPLC and Q-TOF-MS/MS conditions, over 50 peaks were separated and detected in Du–Shen–Tang within 18 min. The components were identified by comparing the mass spectra and retention time with that of reference compounds, and/or tentatively assigned by elucidating low energy CID fragment ions as well as matching empirical molecular formula with that of the published known compounds. Totally 45 major ginsenosides were identified in Du–Shen–Tang, 21 of which were determined to be newly generated during the decoction of ginseng. The mechanisms involved were further deduced to be hydrolysis, dehydration, decarboxylation and addition reactions of the original ginsenosides in white ginseng through analyzing mimic decoctions of 13 pure reference ginsenosides. Significant difference in chemical profiles between decoctions of two batches of white ginseng suggested that storage duration or other factors significantly influenced the quality consistency of not only the crude drug but also the decoction (Du–Shen–Tang) of white ginseng.     

Determination of rat plasma levels of sertraline enantiomers using direct injection with achiral–chiral column switching by LC–ESI/MS/MS

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization–tandem mass spectrometric (2D-LC–ESI/MS/MS) method to determine sertraline (SRT) enantiomers in rat plasma was developed and validated. The method was applied to separate and determine the diastereomers and enantiomers of SRT simultaneously. The 2D-LC–ESI/MS/MS system consisted of RAM column in first dimension for trapping proteineous part of plasma and a chiral Cyclobond column as second dimension for separation of enantiomers and diastereomers of SRT using 0.1% aqueous trifluoroacetic acid:acetonitrile (86:14, v/v) as mobile phase in an isocratic elution mode. The linear dynamic range was 0.5–200 ng/mL (r² > 0.999). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in the analysis of SRT enantiomers in rat plasma to support pharmacokinetic studies.     

Rapid and sensitive analysis of cyclobenzaprine by LC–MS/MS: Application to a pharmacokinetic study of cyclobenzaprine in dog

A rapid and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of cyclobenzaprine in dog plasma. After extracted with organic solvent, post-treatment samples were separated on an Agela C18 column interfaced with a triple quadrupole tandem mass spectrometer in positive electrospray ionization mode. Multiple reaction monitoring was performed using the transitions of m/z 276.2 → 216.1 and m/z 325.1 → 109.0 to quantify cyclobenzaprine and escitalopram (internal standard), respectively. The mobile phase consisted of acetonitrile: 5 mM ammonium acetate: formic acid (90:10:0.01, v/v/v) at a flow rate of 0.3 ml/min. The total analysis time was 2.4 min. The method was linear over the concentration range of 0.0200–10.0 ng/ml. The intra- and inter-day precision was within 12.8% in terms of relative standard deviation (RSD%) and the accuracy within 5.6% in terms of relative error. This method was successfully applied in a pharmacokinetic study of extended-release cyclobenzaprine in dogs.     

Pharmacokinetics of diphenylboroxazolidones of L-α-amino acids with activity on the CNS: quantification in rat DBS by UPLC-MS/MS

Background: A growing number of boron-containing compounds exhibit many important biological activities; of particular interest are the α-amino acid borinic derivatives with activity in the CNS. A validated, sensitive and specific UPLC-MS/MS technique for quantification of the diphenylboroxazolidones of glycine (DBPX-gly), L-aspartate (DPBX-L-asp) and L-glutamate (DPBX-L-glu) in dried blood spots (DBSs) is presented. Results: The most intense signal corresponds to compounds with (11)B. The extraction procedure was liquid elution of 3.2-mm punched DBSs with acetonitrile:aqueous formic acid 0.1% (80:20 v/v). Assays proved to be linear, falling accurately and precisely within the range of 0.3-50 μg/ml for DPBX-L-asp and DPBX-L-glu and 0.1-5 μg/ml for DBPX-gly. Chromatograms exhibit clean 2.0-min running time peaks and S/N ratios for the LLOQ were approximately 15:1. The technique was used to evaluate the pharmacokinetics of the molecules and to correlate these with timecourse toxic effects. Conclusion: DBSs represent an advantage for the collection of small volumes of samples, and also in terms of processing and storage. UPLC-MS/MS allow us not only to identify the isotopic pattern of boron in DBPX, but also to quantify them with accuracy and specificity. Pharmacokinetics of these molecules exhibit a high apparent volume of distribution; it suggests a preference of DPBX-amino acids for fatty tissues such as the CNS.     

Coptis chinensis inflorescence extract protection against ultraviolet-B-induced phototoxicity,and HPLC–MS analysis of its chemical composition

Cultivated Coptis chinensis inflorescence has been highly valued in Chinese tea production for many years. The main alkaloid compounds in C. chinensis inflorescence ethanolic extracts (CE) were identified by high-performance liquid chromatography–mass spectrometry. The detected compounds included jatrorrhizine (4.87 mg/g), coptisine (17.18 mg/g), palmatine (3.32 mg/g), and berberine (31.81 mg/g), as well as columbamine and epiberberine (tentatively identified). CE protective activity against ultraviolet-B (UVB)-induced phototoxicity in a mitochondria model was determined by measuring thiobarbituric acid-reactive substrates, lipid hydroperoxide, conjugated diene, 4-hydroxynonenal, and glutathione. The results showed that CE excellently inhibited UVB-induced lipid peroxidation and glutathione reduction in vitro. This photoprotective effect of CE may be caused by the presence of the abovementioned alkaloid compounds and phenolic compounds that enhances CE antioxidant activity. Therefore, CE possesses potent photoprotective property that may find valuable applications in food industries and in anti-phototoxicity formulations.     

Efficient isolation of catechins from green tea and characterization of interaction property of catechins with proteins by HPLC–UV/DAD combined with ultrafiltration

The aim of this study was to ascertain systematically interaction properties of active compounds in green tea extract (GTE) and involved proteins in the path of drugs delivery by high performance liquid chromatography–UV/DAD combined with ultrafiltration. GTE prepared by 50% methanol was separated successfully into fourteen components under optimized chromatographic conditions. Binding degrees of (?)-catechin (C), (?)-epigallocatechin gallate (EGCG), caffeine, and (?)-epicatechin gallate (ECG) in GTE with human plasma protein, rat plasma protein, human serum albumin (HSA), pancreas membrane protein (PMP), and rat pancreas cell membrane (PCM) were determined, respectively. The binding degrees of C, EGCG, ECG, and caffeine were above 80% of the total plasma (human or rat), and the binding degrees of four components were still above 65% at 0.6?mM HSA solution. The binding degrees of C, EGCG, and ECG with PMP were 36.9, 37.4, and 39.2%, and the binding degrees of EGCG and ECG to PCM were 78.8 and 91.5%, respectively. These results showed that there were high binding degrees between these components and transportation proteins in plasma, and receptor proteins on cell membrane. Compared with C and caffeine, EGCG and ECG had always higher binding degrees with different proteins, which meant EGCG and ECG were true active components. Based on these results, we suggest that the inhibition of the pathogenesis of some diseases by green tea should be associated with the high binding degrees of catechins to target receptors.