Simultaneous quantification of both triterpenoid and steroidal saponins in various Yunnan Baiyao preparations using HPLC–UV and HPLC–MS

Yunnan Baiyao (YNBY) is one of the best known traditional Chinese medicines. Saponins are considered to be its active components. In this study, an HPLC method was first developed for the simultaneous quantitative analysis of thirteen saponins, including five triterpenoid saponins and eight steroidal saponins, in a series of YNBY preparations, i. e., powder, capsules, aerosol, toothpaste, plaster, and adhesive bandage. The pre‐treatment methods for each dosage form were investigated and optimized. The HPLC separation was performed on a Shim‐pack C₁₈ reversed‐phase column in gradient mode with UV detection at 203 nm. All calibration curves showed good linear regression (r² ? 0.9981) within the test ranges. Precisions and repeatabilities of the methods were better than 4.22 and 4.78%, respectively. Recoveries were better than 90.5%, even in the analysis of the least abundant saponins in a complex YNBY plaster. HPLC–ESI‐TOF/MS was used for definite identification of compounds in the preparations. This proposed method was successfully applied to quantify the 13 bioactive constituents in 27 commercial samples to evaluate the quality of YNBY preparations. The overall results demonstrate that this method is simple, reliable, and suitable for the quality control of YNBY. Furthermore, the retention behavior of these saponins in reversed‐phase chromatography is described.     

Analysis of diphenylarsinic acid in human and environmental samples by HPLC–ICP‐MS

A simple, rapid and robust analytical method for determining diphenylarsinic acid in human and environmental samples was developed based on a combination of hydrophilic polymer‐based gel‐permeation high‐performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICP‐MS). Hair and nail samples were digested with alkali, and liberated diphenylarsinic acid (derivative) was extracted with diethyl ether, redissolved in water and injected for HPLC–ICP‐MS analysis. Human urine, groundwater and water extracts from soils were injected for HPLC–ICP‐MS directly after filtration. Using the method, diphenylarsinic acid in a solution was quantified in 7 min duration for an analysis with a detection limit of sub‐nanograms per milliliter. The method has been applied to groundwater arsenic pollution recently uncovered in Japan. Copyright © 2005 John Wiley & Sons, Ltd.     

Determination of organoarsenic species in blood plasma by HPLC–ICP MS

A study of arsenic speciation in blood plasma of patients undergoing renal dialysis has been performed using HPLC coupled with ICP MS. It was found that the only detectable arsenic species present in the plasma was arsenobetaine. The limit of detection using an injection volume of 175 µl was found to be 0.25 ng of arsenic as arsenobetaine. Spiking experiments demonstrated recoveries of approximately 100%. In the absence of certified reference materials or an alternative technique, we believe this was the best way to demonstrate that the method was reliable and accurate. Arsenobetaine concentrations in pre‐dialysis plasma were similar to those for the healthy volunteers, although after dialysis the concentrations were significantly reduced. It is thus concluded that, except for a few patients, dialysis removed the arsenobetaine efficiently (hence preventing an accumulation of arsenic) and that no biotransformations were occurring. The exceptions to this conclusion were in a few patients whose arsenobetaine levels increased marginally after dialysis, but this was attributed to the levels both pre‐ and post‐dialysis being very close to the detection limit. Copyright © 1999 John Wiley & Sons, Ltd.     

Arsenic speciation in some environmental samples: a comparative study of HG–GC–QFAAS and HPLC–ICP–MS methods

Some water and soil extracts polluted with arsenic, and a sewage sludge certified for total arsenic have been analysed by high‐performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC–ICP–MS) and hydride generation–gas chromatography– quartz furnace atomic absorption spectrometry (HG–GC–QFAAS techniques.) Detection limits in the range of 200–400 and 2–10 ng l⁻¹ respectively allowed the determination of inorganic [As(III), As(V)] and methylated (DMA, MMA, TMAO) arsenic species present in these samples. Results obtained by both methods are well correlated overall, whatever the arsenic chemical form and concentration range (8–10 000 μg l⁻¹). Comparison of these results enabled us to point out features and disadvantages of each analytical method and to reach a conclusion that they are suitable for arsenic speciation in these environmental matrices. Copyright © 2000 John Wiley & Sons, Ltd.     

Conformational study on telaprevir by HPLC–DAD–MS and theoretical calculation

Telaprevir is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3‐4A serine protease. it is used for the treatment of HCV infection in combination with peginterferon alfa and ribavirin. In the present work, the E–Z isomerization process of telaprevir in solution was revealed by online HPLC–DAD (diode array detector)–MS, variable‐temperature and variable‐gradient experiments. The molecular geometry information of the two isomers was established by molecular mechanics calculations, and good correlation between the two isomers' UV–vis spectra and their molecular geometry information was also discovered. In addition, it was revealed by molecular docking that the two isomers have different affinities to HCV NS3?4A protease, and the Z isomer, the minor form of telaprevir in solution, is the more effective inhibitor of HCV NS3?4A protease. The investigation can provide more structure information about telaprevir in solution and in the binding process of HCV NS3?4A protease.     

Simple correction improving long‐term reproducibility of HPLC–MS

Chromatographic peak areas in long series of high‐performance liquid chromatography–MS experiments often vary, which decrease reproducibility and may cause bias in the results. It was found that the sensitivity of various components change differently; in our case, variability is in the order of 20–40%, and it is most likely due to changing conditions in electrospray ionization (ESI). The most often used peak area correction methods do not take this effect into account. The change in peak areas can be well described by a polynomial function; we found that a fourth‐order polynomial is most often suitable. We suggest a simple correction algorithm based on polynomial fitting. When the experiments were inherently well reproducible, this correction improved reproducibility from 12% to 3% (on average for various components). When random errors were larger, this improvement was less significant (15% to 12% in nano‐ESI) but nevertheless essential in order to avoid possible bias in the results. Copyright © 2015 John Wiley & Sons, Ltd.     

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC–ESI–IT–TOF–MS

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two‐dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two‐dimensional HPLC–IT–TOF–MS. Molecular docking and 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose‐dependent manner in the range of 5–50 μmol/L. In conclusion, the two‐dimensional EGFR/CMC–HPLC–IT–TOF–MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.     

Simultaneous determination of 19 constituents in Cimicifugae Rhizoma by HPLC–DAD and screening for antioxidants through DPPH free radical scavenging assay

Cimicifugae Rhizoma (sheng ma) is a well‐known traditional Chinese medicine, which has been demonstrated to possess anti‐inflammatory, antipyretic and analgesic activities. In the present study, a simple and efficient HPLC–DAD (high‐performance liquid chromatography coupled with diode array detection) method was developed and validated for simultaneous quantification of 19 chemical components (including 16 phenolic acids, one coumarin and two alkaloids) in Cimicifugae Rhizoma. The result indicated that this method could effectively evaluate the quality of Cimicifugae Rhizoma and provide a valuable reference for further study. Additionally, the antioxidant activity of Cimicifugae Rhizoma was evaluated by DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging assay. The results showed that the content of phenolic acids and antioxidant activity exhibited significant correlation coefficients.     

Use of diffusion‐ordered NMR spectroscopy and HPLC–UV–SPE–NMR to identify undeclared synthetic drugs in medicines illegally sold as phytotherapies

The informal (and/or illegal) e‐commerce of pharmaceutical formulations causes problems that governmental health agencies find hard to control, one of which concerns formulas sold as natural products. The purpose of this work was to explore the advantages and limitations of DOSY and HPLC–UV–SPE–NMR. These techniques were used to identify the components of a formula illegally marketed in Brazil as an herbal medicine possessing anti‐inflammatory and analgesic properties. DOSY was able to detect the major components present at higher concentrations. Complete characterization was achieved using HPLC–UV–SPE–NMR, and 1D and 2D NMR analyses enabled the identification of known synthetic drugs. These were ranitidine and a mixture of orphenadrine citrate, piroxicam, and dexamethasone, which are co‐formulated in a remedy called Rheumazim that is used to relieve severe pain, but it is prohibited in Brazil because of a lack of sufficient pharmacokinetic and pharmacodynamic information. Copyright © 2013 John Wiley & Sons, Ltd.     

Characteristic ion clusters as determinants for the identification of pyrrolizidine alkaloid N‐oxides in pyrrolizidine alkaloid–containing natural products using HPLC–MS analysis

Pyrrolizidine alkaloid (PA)–containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N‐oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC–MS is generally used to analyze PA‐containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC–MS methodology cannot distinguish PA N‐oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N‐oxides and the corresponding PAs were systemically investigated using HPLC–MS to define the characteristic mass fragment ions specific to PAs and PA N‐oxides. Mass spectra of toxic retronecine‐type PA N‐oxides exhibited two characteristic ion clusters at m/z 118–120 and 136–138. These ion clusters were produced by three unique fragmentation pathways of PA N‐oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine‐type PA N‐oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120–122 and 138–140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N‐oxides in two PA‐containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N‐oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N‐oxides from PAs in PA‐containing natural products, which is crucial for the assessment of their intoxication. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of diphenylarsinic acid and phenylarsonic acid,the degradation products of organoarsenic chemical warfare agents,in well water by HPLC–ICP‐MS

Diphenylarsinic acid (DPAA) and phenylarsonic acid (PAA), which were degradation products of organoarsenic chemical warfare agents used as sternutatory gas, were detected in the well water at Kamisu, Ibaraki Prefecture, Japan. The standard material of DPAA was synthesized with aqueous arsenic acid and phenylhydrazine in order to determine organic arsenic compounds in well water. The DPAA showed a protonated ion at m/z 263 [M + H]⁺ and a loss of H₂O ion at m/z 245 [M + H ? H₂O]⁺ from protonated ion by the electrospray ionization time‐of‐flight mass spectrometry. The quantitative analysis of DPAA and PAA was performed by high‐performance liquid chromatography inductively coupled plasma mass spectrometry and the system worked well for limpid liquid samples such as well water. Copyright © 2005 John Wiley & Sons, Ltd.     

Screening and analysis of metabolites in rat urine after oral administration of Apocynum venetum L. extracts using HPLC–TOF‐MS

HPLC with diode array detection and ESI‐TOF‐MS was used for the study of the constituents in Apocynum venetum L. extracts and the metabolites in rat urine after oral administration of A. venetum L. extracts. A formula database of the known constituents in A. venetum L. was established, and 21 constituents were rapidly identified by accurately matching their molecular masses with the formulae of the compounds in the database. Furthermore, 34 metabolites were detected and elucidated in the rat urine. The scientific and plausible biotransformation pathways of the flavonoid components in A. venetum L. were also proposed together with the presentation of clues for potential mechanisms of bioactivity. This specific and sensitive HPLC–ESI‐TOF‐MS method can be used to identify the chemical components in the extracts of A. venetum L. and their metabolites in rat urine. This method can also be used to reveal the possible metabolic mechanisms of action of the extract components in vivo.     

HPLC–MS/MS analysis of peramivir in rat plasma: Elimination of matrix effect using the phospholipid‐removal solid‐phase extraction method

A simple HPLC–MS/MS method has been developed for the determination of peramivir in rat plasma in the present study. The analytes were separated on a C₁₈ column (50 × 2.1 mm, 1.7 μm) and a triple‐quadrupole mass spectrometer equipped with an electrospray ionization source was applied for the detection. A phospholipid‐free cartridge solid‐phase extraction was used to pretreat the plasma and eliminate the endogenous phospholipid. The in‐source collision‐induced dissociation approach showed that this pretreatment could result in negligible ion suppression from the extracted sample and could produce cleaner samples when compared with the protein precipitation. The method was linear over the concentration range of 0.12–1200.0 ng/mL for peramivir. The method was validated and successfully applied to a pharmacokinetic study after peramivir was orally and intravenously administered to Sprague–Dawley rats.     

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC–fluorimetry

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full‐spot analysis of a volumetrically deposited 40 μl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C₁₈ column (5.0 μm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7–acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34–118.89% with recoveries >80%. Bland–Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.     

Investigation of pharmacokinetics,tissue distribution and excretion of schisandrin B in rats by HPLC–MS/MS

Schisandrin B has received much attention owing to its various biological activities. The present study was aimed at the formulation development of schisandrin B and investigation of the pharmacokinetic profiles, distribution and excretion of schisandrin B in Sprague–Dawley rats. In this study, micronized schisandrin B particles with particle size of 10–20 μm were chosen as the research object. Chromatographic separation was carried out on a BDS Hypersil C₁₈ column (50 × 2.1 mm, i.d. 3.5 μm). Schisandrin B and deoxyschizandrin (internal standard) were detected without interference in the multiple reaction monitoring mode with positive electrospray ionization. The pharmacokinetic parameters were calculated by a noncompartmental method. The area under concentration–time curve and the maximum concentration showed a significant difference in gender. The calculated absolute oral bioavailability of schisandrin B was ~55.0% for female rat and 19.3% for male rat. Schisandrin B exhibited linear pharmacokinetics properties within the range of the tested oral dose (10, 20 and 40 mg/kg). After oral administration of schisandrin B, it was extensively distributed in ovary and adipose tissue. The result also showed very low urinary, biliary and fecal excretion of schisandrin B implying that schisandrin B was excreted mainly in the forms of metabolites.     

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC–MS

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC–MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar‐turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose‐dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs.     

Determination of cuticular and internal fatty acids of Chorthippus brunneus males and females using HPLC‐LLSD and GC–MS

Insects are of growing significance in veterinary medicine and human healthcare; therefore, an understanding of their biology is very important. The cuticular and internal fatty acid compositions of Chorthippus brunneus males and females have been studied for the first time. The lipids of males and females were separated into classes of compounds using high‐performance liquid chromatography with a laser light scattering detector. The free fatty acid (FFA) fractions obtained by HPLC were silylated and then analyzed by GC–MS. The cuticular lipids of males contained 15 saturated, four unsaturated with even‐numbered and two unsaturated with odd‐numbered carbon chains, FFAs ranging from C8 to C25. The major free fatty acids in males were C16 (20.8%), C18:2 (8.5%), C18:1 (32.9%) and C18 (24.4%). The cuticular lipids of females contained 17 saturated, four monounsaturated and two diunsaturated free fatty acids ranging from C8 to C30. The major cuticular fatty acids in females were C16 (25.1%), C18:2 (6.2%), C18:1 (23.7%) and C18:0 (33.2%). The internal FFAs of males consisted of 20 compounds ranging from C8 to C26. Four of these compounds were detected as major compounds: C16 (14.1%), C18:2 (21.6%), C18:1 (38.0%) and C18 (22.5%). Among 18 internal free fatty acids of females, C16 (22.3%), C18:2 (10.9%), C18:1 (40.2%) and C18 (20.5%) were the most abundant compounds. The following cuticular fatty acids present in the lipids of females were absent in the lipids of males: C26, C27 and C30. On the other hand, only C24 was absent from the cuticular lipids of females. Only C10 and C24 internal fatty acids present in the lipids of males were absent in the lipids of females. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of abamectin in citrus fruits using SPE combined with dispersive liquid–liquid microextraction and HPLC–UV detection

A new pretreatment method, SPE combined with dispersive liquid–liquid microextraction, was proposed for the determination of abamectin in citrus fruit samples for the first time. In this method, fruit samples were extracted by ultrasound‐assisted extraction followed by SPE. Then, the SPE was used as a disperser solvent in the next dispersive liquid–liquid microextraction step for further purification and enrichment of abamectin. The effects of various parameters on the extraction efficiency of the proposed method were investigated and optimized. Good linearity of abamectin was obtained from 0.005 to 10.0 mg/kg for B_1a and from 0.05 to 10.0 mg/kg for B_1b with correlation coefficient (r²) of 0.998 for B_1a and 0.991 for B_1b, respectively. The LODs were 0.001 and 0.008 mg/kg (S/N = 3) for B_1a and B_1b, respectively. The relative recoveries at three spiked levels were ranged from 87 to 96% with the RSD less than 11% (n = 3). The method has been successfully applied to the determination of abamectin in citrus fruit samples.     

Development of a mild mercaptoethanol extraction method for determination of mercury species in biological samples by HPLC–ICP-MS

A mild, efficient and convenient extraction method of using 2-mercaptoethanol contained extractant solution combined with an incubator shaker for determination of mercury species in biological samples by HPLC–ICP-MS has been developed. The effects of the concentration of 2-mercaptoethanol, the composition of the extractant solution and the shaking time on the efficiency of mercury extraction were evaluated. The optimization experiments indicated that the quantitative extraction of mercury species from biological samples could be achieved by using 0.1% (v/v) HCl, 0.1% (v/v) 2-mercapoethanol and 0.15% (m/v) KCl extractant solution in an incubator shaker for shaking overnight (about 12 h) at room temperature. The established method was validated by analysis of various biological certified reference materials, including NRCC DOLT-3 (dogfish liver), IAEA 436 (tuna fish), IAEA MA-B-3/TM (garfish filet), IAEA MA-M-2/TM (mussel tissue), GBW 08193 (bovine liver) and GBW 08572 (prawn). The analytical results of the reference materials were in good agreement with the certified or reference values of both methyl and total mercury, indicating that no distinguishable transformation between mercury species had occurred during the extraction and determination procedures. The limit of detection (LOD) for methyl (CH₃Hg⁺) and inorganic mercury (Hg²⁺) by the method are both as 0.2 μg L⁻¹. The relative standard deviation (R.S.D.s) for CH₃Hg⁺ and Hg²⁺ are 3.0% and 5.8%, respectively. The advantages of the developed extraction method are that (1) it is easy to operate in HPLC–ICP-MS for mercury species determination since the extracted solution can be directly injected into the HPLC column without pH adjustment and (2) the memory effect of mercury in the ICP-MS measurement system can be reduced.     

The separation of dimethylarsinic acid,methylarsonous acid,methylarsonic acid,arsenate and dimethylarsinous acid on the Hamilton PRP‐X100 anion‐exchange column

In order to separate the potential arsenite metabolites methylarsonous acid and dimethylarsinous acid from arsenite, arsenate, methylarsonic acid and dimethylarsinic acid, the pH‐dependent retention behaviour of all six arsenic compounds was studied on a Hamilton PRP‐X100 anion‐exchange column with 30 mM phosphate buffers (pH 5, 6, 7, 8 and 9) containing 20% (v/v) methanol as mobile phase and employing an inductively coupled plasma atomic emission spectrometer (ICP–AES) as the arsenic‐specific detector. Baseline separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylarsinous acid was achieved with a 30 mmol dm⁻³ phosphate buffer (pH 5)–methanol mixture (80:20, v/v) in 25 min. Arsenite is not baseline‐separated from dimethylarsinic acid under these conditions. Copyright © 1999 John Wiley & Sons, Ltd.