Qualitative and quantitative analysis of traditional Chinese medicine Niu Huang Jie Du Pill using ultra performance liquid chromatography coupled with tunable UV detector and rapid resolution liquid chromatography coupled with time-of-flight tandem mass spectrometry

An ultra performance liquid chromatography coupled with tunable UV detector (UPLC-TUV) and rapid resolution liquid chromatography coupled with time-of-flight tandem mass spectrometry (RRLC-Q-TOF) method was developed for the quality assessment of Niu Huang Jie Du Pill (NHJDP), a commonly used traditional Chinese medicine (TCM). Ten compounds were simultaneously identified by electrospray ion mass spectrometry (ESI/MS) and comparison with reference standards and literature data. All of them were quantified by UPLC method. Baseline separation was achieved on an ODS-140HTP C₁₈ column (2.3 μm, 100 mm × 2.1 mm I.D.) with linear gradient elution of acetonitrile–0.1% formic acid. This developed method provides good linearity (r² > 0.9996), repeatability (RSD < 3.63%), intra- and inter-day precisions (RSD < 0.86%) with accuracies (97.88–101.56%) and recovery (98.88–101.92%) of 10 major constituents, namely baicalin, baicalein, wogonoside, wogonin, glycyrrhizic acid, liquiritin, rhein, emodin, chrysophanol and physcion. In addition, the principal component analysis (PCA) coupled with the UPLC fingerprint was applied to classify the NHJDP samples according to their manufacture corporation. This proposed method with high sensitivity and selectivity was successfully utilized to analyze 10 major bioactive compounds in 30 batches of NHJDPs, and the results demonstrate that this analytical method is simple and suitable for the original discrimination and quality control of this TCM.     

Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector

Bai-Hu-Tang (BHT), a classic traditional Chinese medicine (TCM) formula used for clearing heat and promoting body fluid, consists of four traditional Chinese medicines, i.e., Gypsum Fibrosum (Shigao), Anemarrhenae Rhizoma (Zhimu), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (Zhigancao), and nonglutinous rice (Jingmi). The chemical composition of BHT still remains largely elusive thus far. To qualitatively and quantitatively characterize secondary metabolites and carbohydrates in BHT, here a combination of analytical approaches using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector was developed and validated. A total of 42 secondary metabolites in BHT were tentatively or definitely identified, of which 10 major chemicals were quantified by the extracting ion mode of quadrupole time-of-flight mass spectrometry. Meanwhile, polysaccharides, oligosaccharides, and monosaccharides in BHT were also characterized via sample pretreatment followed by sugar composition analysis. The quantitative results indicated that the determined chemicals accounted for 35.76% of the total extract of BHT, which demonstrated that the study could be instrumental in chemical dissection and quality control of BHT. The research deliverables not only laid the root for further chemical and biological evaluation of BHT, but also provided a comprehensive analytical strategy for chemical characterization of secondary metabolites and carbohydrates in traditional Chinese medicine formulas.     

Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometric procedure for qualitative and quantitative analyses of nortriterpenoids and lignans in the genus Schisandra

An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS) procedure is designed for the first simultaneous analysis of nortriterpenoids and lignans in Schisandra samples. The method consists of three individual mass spectrometric experiments, including the full scan MS, MS/MS experiment and in-source collision induced dissociation (CID) MS/MS, which enable the identification of diagnostic fragmentation pathways of nortriterpenoids and lignans. As such, a total of 6 nortriterpenoids and 10 lignans were unequivocally identified, and one nortriterpenoid and 20 lignans were tentatively identified from different Schisandra samples within 12.5 min. In addition, 6 nortriterpenoids and 10 lignans were quantified in 48 samples of S. chinensis and S. sphenanthera using an extract ion chromatogram (XIC) of the full scan MS experiment. Dataset obtained from UPLC-MS was processed with principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to compare the difference between the two Schisandra species.     

An approach to develop binary chromatographic fingerprints of the total alkaloids from Caulophyllum robustum by high performance liquid chromatography/diode array detector and gas chromatography/mass spectrometry

An approach was proposed to develop binary chromatographic fingerprints by means of high performance liquid chromatography/diode array detector (HPLC/DAD) and gas chromatography/mass spectrometry (GC/MS). HPLC fingerprint and GC/MS fingerprint that, respectively, represent chemical characteristics of aporphinoid and quinolizidine alkaloids of the total alkaloids from Caulophyllum robustum were developed, which were used to construct binary chromatographic fingerprints of the total alkaloids. Moreover, the authentication and validation of the binary fingerprints were performed. Then, a data-level information fusion method was employed to capture the chemical information encoded in two chromatographic fingerprints. Finally, based on the fusion results, the quality of 10 batches of the total alkaloids samples was further evaluated by similarity measure and cluster analysis method. In comparison with conventional fingerprint, the binary chromatographic fingerprints which represent the characteristics of more constitutions can comprehensively and properly reveal the quality characteristics of the total alkaloids. The binary chromatographic fingerprints can reach more objective conclusions in the practice of quality control of the total alkaloids. In conclusion, the binary chromatographic fingerprints are suitable for quality control of the total alkaloids. The presented approach is a powerful and meaningful tool to comprehensively conduct the quality control of traditional Chinese medicine (TCM).     

Simultaneous analysis of flunitrazepam and its major metabolites in human plasma by high performance liquid chromatography tandem mass spectrometry

A sensitive and specific high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC–APCI–MS–MS) method has been developed for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, in human plasma. After the addition of a deuterium labelled internal standard of flunitrazepam, plasma samples were extracted using Oasis® MCX solid phase extraction cartridges. The compounds were separated on a 5 μm Symmetry C18 (Waters) column (3.0×150 mm, i.d.) with a step gradient of acetonitrile-0.1% formic acid at a flow rate of 0.6 ml/min. The overall extraction efficiency was more than 89% for all three compounds. The limits of detection were 0.25 g/l for flunitrazepam, 0.5 μg/l for 7-aminoflunitrazepam, and 2.0 μg/l for N-desmethylflunitrazepam. Within-run accuracies for quality-control samples were between 92.5 and 101.3% of the target concentration, with coefficients of variation <8%. The proposed method enables the unambiguous identification and quantitation of flunitrazepam and its major metabolites in both clinical and forensic specimens.     

Simultaneous determination of salidroside and tyrosol in extracts of Rhodiola L. by microwave assisted extraction and high-performance liquid chromatography

Rhodiola L. has a long history of use in traditional Chinese medicine (TCM) and has shown great promise in recent clinical trials. Salidroside and tyrosol are two important active compounds present in this TCM. In this work, microwave-assisted extraction (MAE) followed by high performance liquid chromatography (HPLC) with a photodiode array detector (DAD) was developed for quantitative analysis of salidroside and tyrosol in Rhodiola L. samples. After systematical investigation, the optimal experimental parameters of soak time (60 min), extraction solvent volume (1g sample, 5 mL), extract solvent composition (50% methanol/water), microwave power (400 W) and extraction time (5 min) were investigated. The optimized method provided satisfactory precision (R.S.D. values less than 7.5%), good recovery (from 94.4 to 123%), and good linear relation in the range of 5.0-500 microg/mL for salidroside and 1.0-100 microg/mL for tyrosol (R2>0.999). The proposed method was applied to quantitative analysis of salidroside and tyrosol in Rhodiola L. samples from five different growing areas. To demonstrate the method feasibility, recirculation was also used to analyze salidroside and tyrosol in Rhodiola L. samples. The proposed MAE-HPLC-DAD is a simple, rapid and low-cost method for quantitative analysis of salidroside and tyrosol, and a potential tool for quality assessment of Rhodiola L. sample.     

The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry

An high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry (HPLC-DAD/MS) based method has been developed for the simultaneous determination of nine coumarin compounds, nodakenin (1), peucedanone (2), marmesin (3), decursinol (4), 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-enyl)coumarin (5), demethylsuberosin (6), decursin (7), decursinol angelate (8) and isoimperatorin (9) in the Korean medicinal herb, Cham-Dang-Gui, the dried root of Angelica gigas (Umbelliferae). The methanol extracts were analyzed by HPLC using a reversed-phase C18 column (5 microm, 4.5 mm x 250 mm) using a gradient acetonitrile-water solvent system at a flow rate of 1.0 ml/min. The analysis of six coumarins (1, 3, 4 and 6-8) with DAD was done at 330 nm and showed excellent linearity (r(2)=0.998-0.999) in a range of 0.2-250 microg/ml for all the compounds. The average recoveries (n=3) were between 96.5% and 110.8%. Identification of each peak was also discussed with the electrospray ionization multi-stage tandem mass spectroscopy (ESI-MS(n)). The amount of these coumarin compounds was evaluated in A. gigas samples. Meanwhile, three coumarins (2, 5 and 9) could not been quantified by DAD because these peaks were overlapped with others. Determination of these compounds could be successfully accomplished with the HPLC-ESI/MS in selected ion monitoring/selected reaction monitoring mode.     

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

大鼠血浆中冬青素A的LC-MS测定法及其药动学研究(英文)

冬青素A系中药毛冬青的主要成分之一, 本文建立了液相色谱质谱法(LC-MS)研究冬青素A在大鼠体内的药动学特征。色谱分离采用C18柱, 甲醇-5 mM 醋酸铵(80:20, v/v) 为流动相质谱检测采用ESI源, 负离子检测, 冬青素A的检测离子为m/z 501.1→501.1,地高辛(内标) 的检测离子为m/z779.4→779.4。大鼠血浆加入磷酸溶液以乙酸乙酯提取, 分取有机层以氮气流吹干, 流动相复溶后进行LC-MS分析。方法学评价表明该法定量限为1.05 ng/mL, 在1.05-525.5 ng/mL范围内线性关系良好。日内和日间变异均小于10%, 提取回收率大于80%。采用建立的LC-MS法进行了大鼠单剂量口服冬青素A后, 其在大鼠体内的药动学研究, 获得了主要的药动学参数。     

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R²X = 0.664, Q² (cum) = 0.622, and PCs = 2) and the supervised partial least square-data analysis (PLS-DA) model (R²X = 0.708, R²Y = 0.461, and Q²Y = 0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.     

Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.     

Identification and control of impurities in streptomycin sulfate by high-performance liquid chromatography coupled with mass detection and corona charged-aerosol detection

For the control of impurities in streptomycin sulfate a reversed phase ion-pair high performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed. With this method, 21 impurities could be separated and tentatively identified using a combination of exact mass measurement by TOF-MS and MS/MS experiments with a triple quadrupole MS. For three impurities the suggested structures could be confirmed by in situ formation. The CAD detector response was found to be linear over 2 orders of magnitude allowing a straightforward quantification of all impurities. A limit of quantification of 0.09% for streptomycin sulfate and of 0.008% for streptidine sulfate (referred to the concentration of the 5 mg/ml test solution) could be achieved. The HPLC method was applied to the purity testing of 12 samples of commercially available streptomycin sulfate from different manufacturers. Impurity levels between 4.6% and 16.0% were found. The current European Pharmacopoeia monograph for streptomycin sulfate only limits streptomycin B by a TLC test to 3.0%. Therefore, the results of this study underline the importance of introducing a state-of-the-art test for the control of impurities in the monograph. The new HPLC-CAD method is considered suitable for this purpose.     

Determination of chlorpromazine in porcine muscle using high performance liquid chromatography-tandem mass spectrometry

A rapid and accurate high performance liquid chromatography tandem mass spectrometry (HPLS-MS) method was established for quantification of chlorpromazine in pork. The porcine samples were pretreated with acetonitrile to precipitate proteins and followed by extraction with tert-butyl methyl ether (TBME). The separation was performed on a 5 µm Agilent XDB-C₁₈ column with gradient elution. The mobile phase A was 0.01 mol/Lammonium formate in water and mobile phase B was acetonitrile. The flow rate was at 0.8 mL/min. Quantification was performed on a triple-quadrupole tandem mass spectrometer using the multiple selected reaction monitoring (MRM) mode. Transition of m/z +319.1 to 58.1 was used for the quantification of chlorpromazine. The method was validated at the concentration range of 0.4040 µg/kgto 8.080 µg/kg for chlorpromazine with correlation coefficient of 0.9999. The spiked recoveries were more than 80.0% and the limit of detection (LOD) was 0.052 µg/kg.The developed method, which offers advantages of convenience, rapid, specificity and higher sensitivity, can be used for determination of chlorpromazine hydrochloride in porcine muscles.     

High performance liquid chromatography (HPLC) fingerprints and primary structure identification of corn peptides by HPLC-diode array detection and HPLC-electrospray ionization tandem mass spectrometry

Corn peptides (CPs) are reported to have many biological functions, such as facilitating alcohol metabolism, antioxidation, antitumor, antihypertension, and hepatoprotection. To develop a method for quality control, the high-performance liquid chromatography (HPLC) system was applied. Twenty-eight common peaks were found in all the CPs of corn samples from Enshi, China, based on which, a fingerprinting chromatogram was established for use in quality control in future research. Subsequently, the major chemical constituents of these common peaks were identified respectively using the HPLC-diode-array detection electrospray ionization tandem mass spectrometry (DAD-ESI-MS/MS) system, and 48 peptide fractions were determined ultimately. This was the first time for the majority of these peptides to be reported, and many of them contained amino acids of glutamine (Q), L and A, which might play an important role in the exhibition of the bioactivities of CPs. Many peptides had a similar primary structure to the peptides which had been proven to be bioactive such as facilitating alcohol metabolism, scavenging free radicals, and inhibiting lipid peroxidation. This systematical analysis of the primary structure of CPs facilitated subsequent studies on the relationship between the structures and functions, and could accelerate holistic research on CPs.     

Metabolism of GTI-2040, a phosphorothioate oligonucleotide antisense, using ion-pair reversed phase high performance liquid chromatography (HPLC) coupled with electrospray ion-trap mass spectrometry

GTI-2040 is a 20-mer phosphorothioate oligonucleotide, which is complementary to the messenger ribonucleic acid (mRNA) of the R2 subunit of ribonucleotide reductase. This study characterized both the in vivo and in vitro metabolism of GTI-2040. A highly specific ion-pair reversed-phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method was used for the identification of GTI-2040 and metabolites from a variety of biological samples including exonuclease enzyme solutions, plasma, urine, mouse liver/kidney homogenates, and human liver microsomes. Progressively chain-shortened metabolites truncated from the 3' terminal of GTI-2040 were detected in all of the evaluated biological samples. GTI-2040 was found to be a good substrate for 3' but not 5' exonuclease. While the pattern of n-1 chain-shortened 3'-exonucleolytic degradation was similar in the mouse liver and kidney homogenates, the latter was found to contain a larger number of shortenmers, the kidneys appeared to possess higher enzymatic reactivity toward GTI-2040. Thus, metabolism of GTI-2040 was found to occur in a variety of biological samples, mainly mediated by the 3' exonuclease.     

Simultaneous determination of dextromethorphan, dextrorphan, and guaifenesin in human plasma using semi-automated liquid/liquid extraction and gradient liquid chromatography tandem mass spectrometry

A method for the simultaneous determination of dextromethorphan (DEX), dextrorphan (DET), and guaifenesin (GG) in human plasma was developed, validated, and applied to determine plasma concentrations of these compounds in samples from six clinical pharmacokinetic (PK) studies. Semi-automated liquid handling systems were used to perform the majority of the sample manipulation including liquid/liquid extraction (LLE) of the analytes from human plasma. Stable-isotope-labeled analogues were utilized as internal standards (ISTDs) for each analyte to facilitate accurate and precise quantification. Extracts were analyzed using gradient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Use of semi-automated LLE with LC-MS/MS proved to be a very rugged and reliable approach for analysis of more than 6200 clinical study samples. The lower limit of quantification was validated at 0.010, 0.010, and 1.0 ng/mL of plasma for DEX, DET, and GG, respectively. Accuracy and precision of quality control (QC) samples for all three analytes met FDA Guidance criteria of +/-15% for average QC accuracy with coefficients of variation less than 15%. Data from the thorough evaluation of the method during development, validation, and application are presented to characterize selectivity, linearity, over-range sample analysis, accuracy, precision, autosampler carry-over, ruggedness, extraction efficiency, ionization suppression, and stability. Pharmacokinetic data are also provided to illustrate improvements in systemic drug and metabolite concentration-time profiles that were achieved by formulation optimization.     

Simultaneous determination of six major stilbenes and flavonoids in Smilax china by high performance liquid chromatography

A simple, sensitive and specific HPLC method was developed for simultaneous determination of the six major active constituents in Smilax china, namely taxifolin-3-O-glycoside (1), piceid (2), oxyresveratrol (3), engeletin (4), resveratrol (5) and scirpusin A (6), respectively. The samples were separated on an Aglient Zorbax XDB-C18 column with gradient elution of acetonitrile and 0.02% phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detected at 300 nm. The six target compounds were completely separated within 35 min. All calibration curves showed good linearity (r2>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.7%. The recoveries were between 93.7 and 103.0%. The method was successfully applied to the analysis of six constituents in 15 commercial samples of S. china. The results indicated that the developed HPLC assay was readily utilized as a quality control method for S. china.     

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.