Phytochemical characterization of bioactive compounds composition of Rosmarinus eriocalyx by RP–HPLC–ESI–QTOF–MS

Rosmarinus eriocalyx (rosemary or Elyazir) is an endemic species growing in arid steppe and rocky mountain in the South-West Algeria. This plant is well known in Algeria and Morocco due to its medicinal properties. However, little is known about its phytochemical composition. For this purpose, natural antioxidant compounds from R. eriocalyx were recovered by solid-liquid extraction and characterized by reversed-phase high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry using negative and positive ionization modes. This analytical methodology enabled the characterization of 101 compounds, which were distributed in five major categories namely hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, flavonoids, phenolic diterpenes and phenolic triterpenes. Moreover, the studied extract generally showed free radical-scavenging and reductive abilities in the range of butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol^, and ascorbic acid. Therefore, the result suggests that the aqueous-methanolic extract of R. eriocalyx could serve as a potential source of antioxidants.     

Quantification of phytochelatins in plants by reversed-phase HPLC–ESI–MS–MS

An on-line HPLC–ESI–MS–MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS–MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 mol kg^–1 plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.Dedicated to the memory of Wilhelm Fresenius     

Investigation of the transformations of a novel anti-cancer agent combining HPLC, HPLC–MS and direct ESI–HRMS analyses

One of the main problems of anti-cancer therapy is an insufficient differentiation between normal and malignant cells by the known anti-proliferant agents. The antibody-directed enzyme prodrug therapy is a promising approach for a selective treatment of cancer, in which a non-toxic prodrug is enzymatically converted into a highly cytotoxic drug at the surface of malignant cells by a targeted antibody–enzyme conjugate. The transformations and the stability of a very promising novel prodrug and its corresponding cytotoxic derivative were now investigated in detail by high-performance liquid chromatography (HPLC)–mass spectrometry (MS). In order to determine the time-dependent DNA alkylation efficiency and the sequence selectivity of the novel compounds, DNA binding studies using direct electrospray–Fourier transform ion cyclotron resonance–MS (ESI–FTICR–MS) have been performed. These measurements were accompanied by HPLC analyses followed by MS of the separated species to confirm the results of the direct ESI–FTICR–MS measurements. The sites of DNA alkylation could be identified unambiguously by the mass spectrometric fragmentation pattern of the alkylated oligodeoxynucleotides as well as by the results of HPLC followed by MS. A combination of all techniques applied led to a better understanding of the mode of action of the new therapeutics and might be used for an estimation of the cytotoxicity of different prodrugs and drugs since the alkylation efficiency correlates with the bioactivity of the compounds in cell culture investigations. After enzymatic cleavage of the sugar moiety, the untoxic prodrug is converted rapidly into the corresponding highly cytotoxic drug that alkylates DNA with high efficiency Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Constituent analysis and quality control of anthocyanin constituents of dried Lycium ruthenicum Murray fruits by HPLC–MS and HPLC–DAD

In recent years, many investigations on the anthocyanins of the fresh Lycium ruthenicum Murray fruits have been reported; while few studies about dried fruits have been published. In this study, chemical profile of dried fruits was illustrated by a high-performance liquid chromatography–tandem mass spectrometry method, which provided evidence for the certain identification of the main anthocyanins. Among these compounds, nine of them were selected as marker compounds for the semiquantitative evaluation, using a simple and reliable method by high-performance liquid chromatography–photodiode array detection (HPLC–DAD), with the combination of chromatographic fingerprint analysis. Separation was achieved on a C18 ODS 80TS QA analytical column with linear gradient elution of acetonitrile and 10% formic acid and 0.1% trifluoroacetic acid (TFA) aqueous solution. Our results showed that the contents of anthocyanins of dried L. ruthenicum Murray from different origins were different. We also inferred the anthocyanin compositions of dried L. ruthenicum Murray through analyzing the UV spectrum, retention time, elution order, and MS data. Finally, eight kinds of anthocyanin compositions were identified and different from the anthocyanins in fresh L. ruthenicum Murray. In a word, this study may provide experimental data in further development and utilization of L. ruthenicum Murray.     

HPLC–DAD–MS analysis of dyes identified in textiles from Mount Athos

Organic colorants contained in 30 textiles (16th to early 20th century) from the monastery of Simonos Petra (Mount Athos) have been investigated using high-performance liquid chromatography equipped with diode-array detection and mass spectrometry (HPLC–DAD–MS). The components of natural dyes identified in samples treated by the standard HCl dyestuff extraction method were: alizarin, apigenin, butein, carminic acid, chrysoeriol, dcII, dcIV, dcVII, ellagic acid, emodin, fisetin, flavokermesic acid, fustin, genistein, haematein derivative (Hae′), indigotin, indirubin, isoliquiritigenin, isorhamnetin, kaempferide, kaempferol, kermesic acid, luteolin, naringenin, purpurin, quercetin, rhamnazin, rhamnetin, sulfuretin, and type B and type C compounds (last two are markers for Caesalpinia trees). Early, semi-synthetic dyes, for example indigo carmine, fuchsin components, and rhodamine B were identified in objects dated late 19th to early 20th century. A dyestuff extraction method which involves use of TFA, instead of HCl, was applied to selected historical samples, showing that the mild method enables efficient extraction of weld (Reseda luteola L.) and dyer’s broom (Genista tinctoria L.) glycosides. The marker compound (Hae′) for logwood (Haematoxylum campechianum L.) identification after treatment with HCl was investigated by liquid chromatography coupled to mass spectrometry (LC–MS) in negative electrospray ionization (LC–MS-ESI⁻) mode. LC–MS in negative atmospheric pressure chemical ionization (LC–MS-APCI⁻) mode was used, probably for the first time, to investigate cochineal (Dactylopius coccus Costa) samples. Positive electrospray ionization (LC–MS-ESI⁺) mode was used for identification of fuchsin components. Detailed HPLC–DAD studies were performed on young fustic (Cotinus coggygria Scop.) and Persian berries (Rhamnus trees).     

Validated HPLC–MS–MS method for determination of azithromycin in human plasma

A validated, highly sensitive, and selective HPLC method with MS–MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na₂EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC–MS–MS. Multiple reaction monitoring mode (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 2.55–551.43 ng mL⁻¹. Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL⁻¹. The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).     

Simultaneous Determination of Key Osmoregulants in Halophytes Using HPLC–ELSD

Osmoregulants are the substances produced by plants that assist in tolerating environmental stresses. Three commonly analysed osomoregulants include mannitol, betaine and proline. A simple, sensitive and rapid HPLC–ELSD method has been developed for the simultaneous analysis of these common osmoregulants in plant extracts. Osmoregulants were extracted using 80 % ethanol and separated on an NH₂ column using 0.1 % formic acid and acetonitrile as the mobile phase. Retention time repeatability was 0.85, 1.50, and 0.93 % for mannitol, betaine and proline, respectively. The limit of detection (μmol) was 1.43 × 10^?4, 7.81 × 10^?5 and 1.08 × 10^?4 for mannitol, betaine and proline, respectively. The developed method was applied to three different plant extracts, Stylosanthes guianensis, Atriplex cinerea and Rhagodia baccata. A second method using a C18 column with 0.1 % heptafluorobutyric acid and acetonitrile as the mobile phase proved to be a useful complementary method for verifying tentative peak identifications.     

Speciation analysis of antimony in extracts of size-classified volcanic ash by HPLC–ICP-MS

Although there is concern about the presence of toxic elements and their species in environmental matrices, for example water, sediment, and soil, speciation analysis of volcanic ash has received little attention. Antimony, in particular, an emerging element of environmental concern, has been less studied than other potentially toxic trace elements. In this context, a study was undertaken to assess the presence of inorganic Sb species in ash emitted from the Copahue volcano (Argentina). Antimony species were extracted from size-classified volcanic ash (<36 microm, 35-45 microm, 45-150 microm, and 150-300 microm) by use of 1 mol L(-1) citrate buffer at pH 5. Antimony(III) and (V) in the extracts were separated and quantified by high-performance liquid chromatography combined on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Antimony species concentrations (microg g(-1)) in the four fractions varied from 0.14 to 0.67 for Sb(III) and from 0.02 to 0.03 for Sb(V). The results reveal, for the first time, the occurrence of both inorganic Sb species in the extractable portion of volcanic ash. Sb(III) was always the predominant species.     

Separation and identification of trinucleotide–melphalan adducts from enzymatically digested DNA using HPLC–ESI–MS

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C₁₈. The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected. Figure Different Alkylation reactions of Melphalan with DNA     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005     

Validation of the RP–HPLC method for analysis of hydrochlorothiazide and captopril in tablets

A rapid and sensitive reverse-phase high performance liquid chromatography (RP–HPLC) method with ultra-violet (UV) detection for a routine control of hydrochlorothiazide and captopril in tablets was developed. The chromatographic system Hewlet Packard 1100 consisted of a HP 1100 pump, HP 1100 UV–VIS detector and HP ChemStation integrator. The samples were introduced through a Rheodyne injector valve with a 20-L sample loop. The isocratic system consisted of a Beckman Ultrasphere ODS 4.6 mm x 15 cm, 5-m-particle column and a mobile phase containing methanol/water (45:55 v/v). The pH of the mobile phase was adjusted to 3.8 with 85% ortophosphoric acid. Quantitation was accomplished using the internal standard method. At the selected conditions, the other excipients of the tablets did not interfere in the assay of active substances. The developed RP–HPLC method was validated, so linearity, precision, accuracy, robustness, limit of quantitation and limit of detection were investigated. For the robustness test, three factors were considered: the composition of the mobile phase , the pH of the mobile phase, and temperature. With the aid of response surface metodology (RSM), it was possible to precisely define the robustness of the method.     

Preparation and Chromatographic Features of Fibrous Core–Shell HPLC Packing Material

In this study, fibrous core–shell silica particles were successfully synthesized via a one-step oil–water biphase stratification coating strategy. The core–shell silica particles were composed of 3-µm non-pore silica cores and thin shells (50–100 nm), which have radial-like direct channels and a large pore size (19.89 nm). The fibrous core–shell silica particles were further modified by n-octadecyltrichlorosilane and used as stationary-phase media in high-performance liquid chromatography (HPLC). The chromatographic properties of the particles were systematically studied in small-molecule and protein separation processes. The results showed that the back pressure was as low as 8.5 MPa under the 1.0-mL min^?1 flow velocity. Furthermore, fibrous core–shell silica particles with an 80-nm shell were used for separating seven small molecules within 10 min and six proteins within 6 min. This work demonstrates that the fibrous core–shell silica particles could be used as an HPLC stationary phase with good performance and low back pressure, and that they have great potential for application to HPLC separation in the future.     

HPLC–UV–ESI MS/MS identification of the color constituents of sawwort (Serratula tinctoria L.)

Extracts from wool dyed with sawwort (Serratula tinctoria L.) obtained with methanol/formic acid and methanol/hydrochloric acid solutions were examined by high-performance liquid chromatography with UV detection coupled with electrospray ionization tandem mass spectrometry. Chromatograms and mass spectra were registered in the negative ion mode under various orifice voltages and collision energies, which enabled us to observe signals corresponding to [M???H]^? ions and also Y^? and/or Y^?? ions, which were further subjected to fragmentation. The results obtained allowed us to define previously unknown constituents of sawwort, which are proposed as specific markers for its identification: chlorogenic acid and its isomers, luteolin-O-glucuronides, eriodictyol-O-glucuronides, and diosmetin-O-glucuronides. Moreover, it was found that during extraction, flavonoid O-glucuronides react with methanol in the presence of hydrochloric acid, forming stable O-methylated derivatives.     

Identification of selenium species in urine by ion-pairing HPLC–ICP–MS using laboratory-synthesized standards

This study focused on the detection/identification of possible selenium metabolites in human urine. Organoselenium compounds not commercially unavailable were synthesized and characterized by electrospray mass spectrometry. Separation of selenomethionine, methylselenomethionine, trimethylselonium, selenoethionine, and selenoadenosylmethionine was achieved by ion-pairing HPLC with a mobile phase of 2 mmol L^–1 hexanesulfonic acid, 0.4% acetic acid, 0.2% triethanolamine (pH 2.5), and 5% methanol. The column effluent was introduced on-line to inductively coupled plasma–mass spectrometry for selenium-specific detection (⁷⁷Se and ⁷⁸Se). For selenium speciation in urine, solid-phase extraction was carried out using C₁₈ cartridges modified with hexanesulfonic acid. Selective retention of cationic species was observed from acidified urine (perchloric acid, pH 2.0). After elution with methanol, evaporation, and dissolution in the mobile phase, the sample was introduced to the HPLC–ICP–MS system and the chromatographic peaks were assigned by adding standards. The species identified in urine were selenomethionine, trimethylselonium ion, and selenoadenosylmethionine. The last species was detected for the first time and our results suggest that selenomethionine might enter the metabolic pathway of its sulfur analog in the activated methylation cycle.Kazimierz Wrobel and Katarzyna Wrobel are on the leave from the Institute of Scientific Research, University of Guanajuato, L. de Retana No. 5, 36000 Guanajuato, Gto., Mexico     

Development of Stability Indicating HPLC–UV Method for Determination of Daclatasvir and Characterization of Forced Degradation Products

A simple and sensitive stability indicating high performance liquid chromatography method was developed for quantification of Daclatasvir hydrochloride in bulk and tablet dosage forms. The analysis was performed on water symmetry analytical column (150 mm?×?3.9 mm, 5 µm), packing octyl silica (Si-[CH₂]₇-CH₃) C8. Mobile phase containing potassium phosphate buffer (pH 2.0) and acetonitrile (38: 62) v/v was used at flow rate 0.7 mL min^?1 for isocratic elution. Detection was performed on 304 nm using UV detector. The method was validated appropriately according to the requirements of United State Pharmacopeia and International Conference on Harmonization guideline Q2 (R1). Recovery, precision, linearity and specificity of the method were assured. The correlation coefficient for linearity ranged from 2 to 24 µg mL^?1 was (r?>?0.9999). The limits of detection and quantification of Daclatasvir were 0.08 and 0.28 µg mL^?1, respectively. Stability studies of Daclatasvir were performed under various stressed conditions, i.e., hydrolytic (acidic, basic and neutral), oxidation, photolytic and thermal conditions, according to International Conference on Harmonization Q1A (R2) and QIB Guidelines. The degradation products were resolved using proposed method and further characterized by MS, NMR and IR spectroscopic analyses. The proposed method was successfully applied to assay determination of bulk drugs and tablet dosage forms.     

Analysis of pesticide residues in essential oils of citrus fruit by GC–MS and HPLC–MS after solid-phase extraction

A robust reliable method for the analysis of residues of pesticides in citrus groves was developed. Residues of twelve pesticides were extracted from citrus essential oils by SPE, separated by liquid chromatography and analyzed by GC-MS. In addition, ten pesticides were extracted by SPE, separated and analyzed by electrospray HPLC-MS. In the case of lemon essential oils, all twenty residues were separated by liquid/solid extraction on a mixed Florisil-C(18) cartridge. The method enabled the analysis of the twenty pesticide residues at levels of 2 to 30 ppm with limits of detection ranging between 0.02 to 0.50 mg L(-1).     

Gamma radiolytic degradation of fluoranthene and monitoring of radiolytic products using GC–MS and HPLC

Removal of priority pollutant fluoranthene in methanol by gamma-irradiation under varied conditions has been optimized. The influence of applied dose and dose rate on the degradation of fluoranthene under nitrogen has been investigated. The preliminary radiolytic degradation efficiency has been monitored by spectrophotometry. HPLC and GC–MS have been used to study the nature of degradation pattern. It is found that four main degradation products are formed and detected by HPLC. Different reversed phase columns have been used for the separation of degraded products under optimum chromatographic conditions. For 2 kGy dose ⩾80% fluoranthene has been degraded at dose rate 200 Gy/h. However, a dose of 370 Gy/h was more effective and it produces for less degradation products. Radiolytic degraded fluoranthene was also analyzed to detect various degradation products using GC–MS. It was proposed that major products were hydrocarbons and methoxy group containing organic compounds after comparing their mass spectra with the installed NIST mass spectral library.     

Novel separation method for highly sensitive speciation of cancerostatic platinum compounds by HPLC–ICP–MS

A high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP–MS) method is presented for analysis of cisplatin, monoaquacisplatin, diaquacisplatin, carboplatin, and oxaliplatin in biological and environmental samples. Chromatographic separation was achieved on pentafluorophenylpropyl-functionalized silica gel. For cisplatin, carboplatin, and oxaliplatin limits of detection of 0.09, 0.10, and 0.15 g L^–1, respectively, were calculated at m/z 194, using aqueous standard solutions. (3 L injection volume). The method was utilized for model experiments studying the stability of carboplatin and oxaliplatin at different chloride concentrations simulating wastewater and surface water conditions. It was found that a high fraction of carboplatin is stable in ultrapure water and in solutions containing 1.5 mol L^–1 Cl^–, whereas oxaliplatin degradation was increased by increasing the chloride concentration. In order to support the assessment of oxaliplatin eco-toxicology, the method was tested for speciation of patient urine. The urine sample contained more than 17 different reaction products, which demonstrates the extensive biotransformation of the compound. In a second step of the study the method was successfully evaluated for monitoring cancerostatic platinum compounds in hospital waste water.     

Simultaneous Determination of Toxic Alkaloids in Blood and Urine by HPLC–ESI–MS/MS

A sensitive and selective method for simultaneous determination of 29 toxic alkaloids in human blood and 31 in urine using high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry was developed and validated. The samples were diluted with 0.1 mol L⁻¹ HCl, and the target alkaloids were purified by solid phase extraction. The separation of 31 alkaloids was carried out on a C18 column using a gradient mobile phase with 10 mmol L⁻¹ ammonium formate in water with 0.1% formic acid and methanol at the rate of 0.25 mL min⁻¹. The triple-quadrupole mass spectrometer equipped with an electrospray source in the positive mode was set up in the dynamic multiple reactions monitoring mode (dynamic MRM) to detect the ion transitions of 31 alkaloids. The calibration curves were linear over a range of 0.5–400, 1–400, or 4–400 μg L⁻¹ for target alkaloids in human blood and urine, and the correlation coefficients (r ²) was higher than 0.9943. The limit of determination and limit of quantification were 0.2–1 and 0.5–4 μg L⁻¹ for blood and urine, respectively. The only exceptions were sanguinarine and chelerythrine in human blood. All the target alkaloids were stable under the test condition. In addition, the solvent effect and reconstituted solution were investigated. The method was validated and proved to be accurate and precise over the studied concentrations and suitable for poisoning diagnosis and forensic toxicology.