Simultaneous quantification of 14 bioactive constituents in Forsythia Suspensa by liquid chromatography–electrospray ionisation–mass spectrometry

Introduction – Forsythia suspensa is a commonly used traditional Chinese medicine including phenylethanoid glycosides, lignans, flavonoids, terpenes and volatile oils. Quantification of multi‐components is important for the quality control of Forsythia suspensa. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous quantification of 14 bioactive constituents of Forsythia suspensa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a Kromasil C₁₈ column (150 ¥ 4.6 mm, 5 mm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. Detection was performed in negative ionisation mode by monitoring the precursor–product combination in multiple reaction monitoring (MRM) mode. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. Results – All calibration curves showed good linear regression (r > 0.9990) within test ranges. The established method showed good precision and accuracy with overall intra‐day and inter‐day variations of 0.7–4.3 and 1.1–3.9% respectively, and overall recoveries of 96.65–101.2% for the compounds analysed. Conclusion – The proposed method was successfully applied for the quantitative analysis of 14 constituents in 12 Forsythia suspensa samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of lanostane triterpenes in Fomes officinalis and their fragmentation study by HPLC-ESI

Introduction – The fruit bodies of Fomes officinalis are used for the treatment of coughs, gastric cancer, rheumatism and hydropsia; however, no method is currently available to assess the quality of this medicinal fungus based on quantitative profile of its main triterpenes. Objective – To develop a simple and accurate HPLC‐UV method for the simultaneous quantification of five lanostane‐type triterpenes in the fruit bodies of F. officinalis. Method – Separations were performed on an Agilent Zorbax Eclipse XDB‐C₁₈ column by gradient elution using acetonitrile : formic acid. Analytes were identified by HPLC coupled with electrospray ionisation mass spectrometry experiments. The quantitative HPLC‐UV method was validated for linearity, precision, accuracy and limits of detection and quantification. Results – Calibration curves presented good linear regression (r > 0.9996) within test ranges. The relative standard deviation of this method was less than 1.7% for intra‐ and inter‐day assays and overall recoveries were 96.4–104.1% for the five compounds analysed. The method was successfully applied to the quantification of five triterpenes in 16 samples of F. officinalis collected from different regions. Conclusion – The developed assay could be considered as a suitable quality control method for F. officinalis. Copyright © 2010 John Wiley & Sons, Ltd.     

A validated liquid chromatography–electrospray ionization–mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr.

Introduction – The medicinal plant Spilanthes acmella (L.) Murr. has demonstrated an array of biological activities that are generally attributed to the presence of spilanthol and other alkylamides. Recently this plant has been of interest due to its potential for the treatment and prevention of malaria. Objective – The aim of this study was to develop a liquid chromatography–electrospray ionisation–mass spectrometry (HPLC‐esiMS) method for rapid identification and quantification of the alkylamide spilanthol from S. acmella. Methodology – Hydroethanolic extracts were prepared from fresh S. acmella using different percentages of ethanol and were stored at ?80, ?20 and 25°C. Spilanthol was isolated and used as a standard for quantitative analysis. Results – Validation parameters for the HPLC‐esiMS analysis of spilanthol were as follows: repeatability, ≤6%; intermediate precision, ≤2%; range, 0.45–450 µm ; limit of detection, 0.27 µm ; and limit of quantification, 0.45 µm . Eight alkylamides in the S. acmella extract were identified based on MS‐MS fragmentation patterns, and NMR analysis confirmed the identity of the most abundant of these as spilanthol. Spilanthol was extracted most efficiently in solvents containing >75% ethanol, and was stable in ethanolic extracts stored at all three temperatures. Conclusion – These results demonstrate the effectiveness of HPLC‐esiMS for quantitative and qualitative analysis of spilanthol. We show that spilanthol is effectively extracted in ethanol, and is stable in ethanol extracts for over 6 months, even at room temperature. Copyright © 2010 John Wiley & Sons, Ltd.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva‐ursi extracts

Introduction – Arbutin is a skin‐whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin‐whitening products by HPLC. Objective – To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva‐ursi extracts. Methodology – N,O‐Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB‐5 narrow bore column. GC‐MS was used for the compound identification, and the quantification was carried out by GC‐FID. The quantitative results were compared with those of HPLC analysis. Results – The developed method gave a good sensitivity with linearity in the range 0.33–500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva‐ursi extracts. The relative standard deviations (RSD) relating to intra‐day and inter‐day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. Conclusion – The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantiospecific Analysis of 8‐Prenylnaringenin in Biological Fluids by Liquid‐Chromatography‐Electrospray Ionization Mass Spectrometry: Application to Preclinical Pharmacokinetic Investigations

8‐Prenylnaringenin (8PN) is a naturally occurring bioactive chiral prenylflavonoid found most commonly in the female flowers of hops (Humulus lupulus L.). A stereospecific method of analysis for 8PN in biological fluids is necessary to study the pharmacokinetic disposition of each enantiomer. A novel and simple liquid chromatographic‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) method was developed for the simultaneous determination of R‐ and S‐8PN in rat serum and urine. Carbamazepine was used as the internal standard (IS). Enantiomeric resolution of 8PN was achieved on a Chiralpak^® AD‐RH column with an isocratic mobile phase consisting of 2‐propanol and 10 mM ammonium formate (pH 8.5) (40:60, v/v) and a flow rate of 0.7 mL/min. Detection was achieved using negative selective ion monitoring (SIM) of 8PN at m/z 339.15 for both enantiomers and positive SIM m/z at 237.15 for the IS. The calibration curves for urine were linear over a range of 0.01–75 µg/mL and 0.05–75 µg/mL for serum with a limit of quantification of 0.05 µg/mL in serum and 0.01 µg/mL in urine. The method was successfully validated showing that it was sensitive, reproducible, and accurate for enantiospecific quantification of 8PN in biological matrices. The assay was successfully applied to a preliminary study of 8PN enantiomers in rat. Chirality 26:419–426, 2014. © 2014 Wiley Periodicals, Inc.     

Quantitative analysis of safranal in saffron extract and nanoparticle formulation by a validated high‐performance thin‐layer chromatographic method†

Introduction – Safranal is an effective anticonvulsant shown to act as an agonist at GABA_A receptors. Nose to brain delivery via nanoparticle formulation might improve its brain delivery. A selective and sensitive analytical method is required for evaluation of safranal‐based novel drug delivery systems. Objective – To develop and validate a high‐performance thin‐layer chromatographic (HPTLC) method for the quantitative analysis of safranal as bulk, in saffron extract and in developed safranal‐loaded nanoparticle formulation. Methodology – Chromatographic separation was achieved on silica gel pre‐coated TLC aluminium plates 60F‐254, using n‐hexane:ethyl acetate (9 : 1, v/v) as the mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 310 nm. The method was validated and applied to detect related impurities, to analyse safranal in saffron extract and to evaluate safranal‐loaded nanoparticles. Results – Compact spots of safranal were observed at R_f value 0.51 ± 0.02. The method was linear (r = 0.9991) between 0.5 and 5.0 μg/spot. The intra‐ and inter‐day precisions were 1.08–2.17 and 1. 86–3.47%, respectively. The limit of detection was 50 ng/spot and the limit of quantification was 150 ng/spot. The method proved to be accurate (recovery 97.4–102.0%) and was selective for safranal. Evaluation of safranal‐loaded nanoparticle formulation demonstrated drug loading of 23.0%, encapsulation efficiency of 42.0% and sustained drug release following biphasic pattern. Conclusion – The present method is useful for the quantitative and qualitative analysis of safranal and safranal‐loaded nanoparticle formulation. It provides significant advantages in terms of greater specificity and rapid analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of lysergide in urine by high-performance liquid chromatography combined with electrospray ionisation mass spectrometry

A quantitative method which avoids derivatisation is described for the determination of lysergide (LSD) levels in urine. Sample preparation included addition of methysergide as an internal standard followed by solid-phase extraction. LSD was analysed on a system consisting of a C₁₈ stationary phase and a mobile phase of 0.1 M acetate buffer pH 8.0-acetonitrile-triethylamine (75:25:0.25, v/v). LSD was detected by electrospray ionisation mass spectrometry with selected ion monitoring. The quantification limit was 0.5 ng/ml and the method was linear up to 10 ng/ml of LSD in urine.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination and pharmacokinetic study of chiisanogenin in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry

Introduction – Chiisanogenin existing in many Acanthopanax species has been reported to possess anti‐inflammatory, antibacterial and antiplatelet aggregatory activities. Objective – To develop and validate a rapid and sensitive ultra performance liquid chromatography‐tandem mass spectrometry method for the determination of chiisanogenin in rat plasma and to investigate its pharmacokinetics after oral administration of chiisanogenin or the extract of Acanthopanax sessiliflorus fruits. Methodology – The sample pretreatment involved a one‐step extraction of 0.2 mL plasma with diethyl ether. Acetaminophen was used as the internal standard. The separation was carried out on an ACQUITY UPLC? BEH C₁₈ column with a mobile phase of acetonitrile‐5 mM ammonium acetate (90:10, v/v) at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Results – A high sample throughput was achieved with an analysis time of 1.1 min per sample. The calibration curve was linear (r² ≥ 0.99) over the concentration range of 5–500 ng/mL with a lower limit of quantification (LLOQ) of 5 ng/mL. The intra‐day and inter‐day precision (relative standard deviation, R.S.D.) values were below 11% and the accuracy (relative error, R.E.) was within 8% at all three quality control (QC) levels. Conclusion – The method was successfully applied to the pharmacokinetic study of chiisanogenin in rat after oral administration of chiisanogenin and the extract of Acanthopanax sessiliflorus fruits. Other constituents in the extract affected the pharmacokinetic behavior of chiisanogenin. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe‐emodine,emodine, rheine,chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae)

Introduction – Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the flora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of different methods of extraction. Objective – After a partial validation of an HPLC method for the simultaneous determination of five anthraquinones, aloe‐emodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three different methods of extraction. Methodology – Anthraquinones were extracted from the bark of R. alpinus using different techniques (methanol maceration, ultrasonic and supercritical CO₂ extraction). Separation and quantification of anthraquinones were accomplished using a reversed‐phase C₁₈ column with the mobile phase of H₂O–methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm. Results – All calibration curves were linear over the concentration range tested (10–200 mM) with the determination coefficients ≥0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All five anthraquinones were found in the samples tested at concentrations reported in experimental data. Conclusion – The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantification of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of polyphenolics in extracts of Potentilla species by high‐performance thin‐layer chromatography photodensitometry method

Introduction – Separation of polyphenolics in different plant materials using high‐performance thin‐layer chromatography (HPTLC) represents an effective method for their detection and quantification. Objective – To develop a simple, specific, precise, sensitive and accurate method for the simultaneous quantification of tiliroside (TRS), methyl brevifolincarboxylate (MBR) and ellagic acid (EA) in a plant extract using the HPTLC‐photodensitometry method. Methodology – Aerial parts of the selected Potentilla species, P. anserina, P. erecta, P. grandiflora and P. nepalensis var. ‘Miss Willmott’, were extracted with methanol. After solvent evaporation, the methanolic extracts were diluted with water and successively partitioned between chloroform and then diethyl ether. The diethyl ether extracts from each sample were used for quantification. The analyses were performed on HPTLC precoated silica gel 60F₂₅₄ plates with toluene–ethyl formate–formic acid (6 : 4 : 1 v/v/v) as the mobile phase (distance of 7.5 cm). Densitometric detections of TRS, MBR and EA were performed at 320, 287 and 280 nm, respectively. The amounts of these compounds were calculated using the regression equations of the calibration curves, which were linear within a range of 0.05–0.5 μg/spot (R² = 0.9957) for TRS, 0.05–0.525 μg/spot (R² = 0.9965) for MBR and 0.0525–0.5 μg/spot (R² = 0.9998) for EA. Results – The amounts of marker compounds measured by the method developed are expressed in mg/g of dry extracts. TRS ranged from 20.3 ± 0.3 mg/g for P. erecta herbs to 197.7 ± 2.9 mg/g for P. grandiflora herbs; MBR ranged from 5.0 ± 0.6 mg/g for P. erecta herbs to 68.5 ± 3.4 mg/g for P. nepalensis flowers; and EA ranged from 24.0 ± 0.6 mg/g for P. erecta herbs to 216.2 ± 3.2 mg/g for P. anserina leaves. Conclusion – The proposed method was found to be relatively simple, specific, precise, sensitive and accurate and may be used for the routine assay of simultaneous determination of TRS, MBR and EA in other extracts and phytomedicines containing Potentilla species. Copyright © 2009 John Wiley & Sons, Ltd.     

Composition and Chemical Variability of Cleistopholis patens Trunk Bark Oil from Côte d'Ivoire

The chemical composition of trunk bark oil from Cleistopholis patens (Benth .) Engl . & Diels , growing wild in Côte d'Ivoire, has been investigated by GC (FID) in combination with retention indices, GC/MS and ¹³C‐NMR. Moreover, one oil sample has been subjected to CC and all the fractions analyzed by GC (RI) and ¹³C‐NMR. In total, 61 components have been identified, including various sesquiterpene esters scarcely found in essential oils. ¹³C‐NMR was particularly efficient for the identification of a component not eluted on GC and for the quantification of heat‐sensitive compounds. Then, 36 oil samples, isolated from trunk bark harvested in six Ivoirian forests have been analyzed. The content of the main components varied drastically from sample to sample: (E)‐β‐caryophyllene (0.4 – 69.1%), β‐pinene (0 – 57%), α‐phellandrene (0 – 33.2%), α‐pinene (0.1 – 30.6%), β‐elemol (0.1 – 29.9%), germacrene D (0 – 25.4%), juvenile hormone III (0 – 22.9%), germacrene B (0 – 20.6%) and sabinene (tr‐20.3%). Statistical analysis, hierarchical clustering and principal components analysis, carried out on the 36 compositions evidenced a fair chemical variability of the stem bark oil of this species. Indeed, three clusters have been distinguished: the composition of group I (ten samples) was dominated by β‐pinene and α‐pinene, group II (nine samples) was represented by α‐phellandrene and p‐cymene and group III (16 samples) by β‐elemol. A sample displayed an atypical composition dominated by (E)‐β‐caryophyllene.     

Enantioselective LC/ESI-MS/MS analysis and pharmacokinetic and tissue distribution study of (2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol in rats

A sensitive and stereospecific liquid chromatography‐tandem mass spectrometry method for the quantitative determination of TWo8 enantiomers ((2RS)‐1‐(7‐methoxy‐1H‐indol‐4‐yloxy)‐3‐(2‐(2‐methoxyphenoxy)ethylamino)‐propan‐2‐ol) was developed and validated in rat serum and some tissues. Racemic TWo8 is a new chemical entity, and it has been shown to possess pharmacological activity in vivo. The assay involved the diastereomeric derivatization of racemic TWo8 with 2,3,4,6‐tetra‐O‐acetyl‐beta‐glucopyranosyl isothiocyanate. The TWo8 diastereoisomers quantification was performed on a triple quadrupole mass spectrometer employing an electrospray ionization technique. The precursor to the product ion transition for TWo8 derivatives and for the internal standard (carbamazepine) was m/z 776.4 → 387.2 and 237.4 → 194.4, respectively. The assay was validated with a linear range of 10–2000 ng/ml of racemic TWo8. The inter‐day precisions for (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 were 2.1% to 14.9% and 1.3% to 14.8%, respectively. The inter‐day accuracy for (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 was within 86% to 114% and 91% to 114%, respectively. A pilot pharmacokinetic study of this new β‐adrenolytic compound has shown that (?)‐(S)‐TWo8 is eliminated faster than its antipode. The terminal half‐lives of (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 were 3.2 and 3.9 h, respectively. The compound distribution into different organs, evaluated in tissue homogenate samples following TWo8 intravenous administration, showed an enantioselective penetration of TWo8 enantiomers in the liver (p < 0.03), in the kidney (p < 0.001), and in the lungs (p < 0.05). The developed method using liquid chromatography‐tandem mass spectrometry method with electrospray ionization could be employed for quantitative determination of compounds with similar structure. Chirality 24:591–599, 2012. © 2012 Wiley Periodicals, Inc.     

Quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR

Aim: To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR. Methods and Results: A real‐time PCR including a calibrator based on plasmid DNA for quantification by means of ΔΔCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 10¹ and 10⁷ copies per μl reaction volume. The linearity calculated by logistic regression revealed a slope of ?3·329 reflecting an efficiency of 99·7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome. Conclusion: The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples. Significance and Impact of the Study: In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real‐time PCR enhances a reliable quantification and is even more sensitive.     

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography–circular dichroism (LC‐CD) method was developed for the simple and rapid identification of N‐octylnortadalafil [(6R, 12aR)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione; RR‐OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC‐CD using Chiralcel OD‐RH (4.6 × 1 50 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR‐OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR‐OTDF and (6R, 12aS)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione were detected in a dietary supplement. Chirality 28:204–208, 2016. © 2016 Wiley Periodicals, Inc.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.