Validation of an LC–MS/MS assay of terpene trilactones in Ginkgo biloba extracts and pharmaceutical formulations through standard addition method

A chromatographic column packed with 1.8 μm particle size octadecyl modified silicagel was used to separate terpene trilactones from Ginkgo biloba extracts/pharmaceutical formulations. Gradient elution was applied, using acidic methanol and water as mobile phase components (0.1% formic acid addition). No specific sample preparation is needed, except dissolution/extraction in methanol of the solid material. Baseline separation of bilobalide and ginkgolides A, B, C and J is obtained within 4 min. The gradient profile is needed to elute the remaining matrix from column. A separation cycle takes 7 min, including column re-equilibration. MS/MS detection with positive electrospray ionization and triple quadrupole mass analysis was used. Multiple reaction monitoring mode was applied for data acquisition, taking protonated molecular ions as precursors. In order to generate reproducible ionization conditions, the standard addition method was considered. The assay of terpene trilactones obtained under such conditions was validated according to guidances in place. Method intermediate reproducibility corresponds to a relative standard deviation of less than 10%. Accuracy, expressed in terms of absolute percent bias ranged from 90% to 110%. Spectral confirmation of target analytes was also included in the validation procedure.     

A fast and efficient LC–MS/MS method for detection,identification and quantitative analysis of bioactive sesterterpenes in Salvia dominica crude extracts

Sesterterpenes are a small group of terpenoids showing a number of interesting pharmacological properties, including cytotoxicity, anti-inflammatory, anti-microbial and anti-angiogenic activities and platelet aggregation inhibition. Recently, some sesterterpene lactones isolated from Salvia dominica have been shown to modulate enzymatic activity of tubulin tyrosine ligase (TTL), a promising target for new anticancer therapeutic strategies. However, to allow a direct use of S. dominica extracts as a source of TTL inhibitors, analytical method aimed to their fast qualitative and quantitative characterization is required. Despite the structural features and diverse biological activities of sesterterpenoids, actually no analytical method for their quantization into complex mixtures has been published. Here we describe an LC–MS/MS method aimed to qualitative and quantitative analysis of sesterterpenes lactones in the crude extracts obtained from different parts of S. dominica. This approach allowed us to characterize all the sesterterpenes by a single step analysis and also to identify two unknown compounds. Moreover, a quantitative comparison of the composition in sesterterpenes of extracts obtained from S. dominica leaves, roots and leaf galls was performed, leading to the definition of both leaves and leaf galls as suitable sources of TTL inhibitors.     

GC–MS profiling of the phytochemical constituents of the oleoresin from Copaifera langsdorffii Desf. and a preliminary in vivo evaluation of its antipsoriatic effect

Copaiba is the oleoresin (OR) obtained from Copaifera (Fabaceae), a neotropical tree which grows in Amazon regions. The balsam, constituted by an essential oil and a resinous fraction is used as folkloristic remedy in the treatment of several inflammatory diseases and for its antioxidant and antibacterial properties. Aim of this work was (a) to carry out a characterization by GC–MS of the volatile and nonvolatile constituents of Copaifera langsdorffii Desf. oleoresin (OR); (b) to investigate the mechanism of its anti-inflammatory activity; (c) to evaluate its antipsoriatic effect after oral intake/topical application. The volatile fraction (yield: 22.51%, w/w) shows: α-bergamotene (48.38%), α-himachalene (11.17%), β-selinene (5.00%) and β-caryophyllene (5.47%). The OR residue (77.49%, w/w), after derivatization, showed as main constituents the following compounds: copalic, abietic, daniellic, lambertinic, labd-7-en-15-oic, pimaric, isopimaric acids and kaur16-en18-oic acid.     

GC–MS analysis of liposoluble constituents from the stems of Cynomorium songaricum

In order to evaluate the differences and similarities between the liposoluble constituents in Cynomorium songaricum populations, stem liposoluble constituents in five populations of C. songaricum collected from three different geographic regions and four different hosts were obtained by solvent extraction and analyzed by GC–MS. Cluster analysis of the percentage composition of 80 compounds showed differences in chemical composition which were related to the geographic origin rather than the host. Hexadecanoic acid was the most abundant compound in the essential oils of C. songaricum from hosts Nitraria sibirica and Nitraria tanguticum. Whereas (Z)-9-octadecenoic acid was accumulated in the oils of C. songaricum from Zygophyllum xanthoxylum and Peganum harmala. Four of the five populations had characteristic components, which were specific to each population.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

LC–MS/TOF and UHPLC–MS/MS study of in vivo fate of rifamycin isonicotinyl hydrazone formed on oral co-administration of rifampicin and isoniazid

The formation and fate of 3-formylrifamycin isonicotinyl hydrazone (HYD) was investigated following oral co-administration of rifampicin (RIF) and isoniazid (INH) in Sprague–Dawley (SD) rats (n = 5) using advanced analytical modalities. The study was carried out with 20 and 5 mg/kg doses of RIF and INH, respectively. The plasma, urine and faeces samples were collected at different time points up to 48 h, which were qualitatively and quantitatively evaluated for the presence of HYD after proper sample preparation. For the same, initially liquid chromatography–mass spectrometry/time-of-flight (LC–MS/TOF) method was developed in electrospray ionization (ESI) positive mode, wherein separation was achieved on a C18 column (4.6 mm × 250 mm, 5 μm), using a volatile mobile phase in a gradient mode. The presence of HYD was confirmed by accurate mass study, spiking with the standard and UV–visible spectra matching. For quantitative evaluation of HYD, a selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method was developed for all the three matrices. In this case, elution of HYD was achieved on a small C18 column (4.6 mm × 50 mm, 1.8 μm) using a short gradient method. The quantitation was done by selective reaction monitoring (SRM) in ESI positive mode. The validation parameters like linearity, accuracy, precision, selectivity, matrix effect, recovery and stability were assessed as per regulatory guidelines. The calibration range was established between 1 and 200 ng/ml, with r² > 0.99 in all the cases. The back calculated values for three quality control (QC) samples, and at lower limit of quantitation (LLOQ) were within 15 and 20%, respectively, of the nominal values. Similarly, the intra- and inter-day precisions were found within 15% at the four tested levels. The HYD was found to be stable for the duration of sample preparation and analysis in the controlled experimental conditions. The analysis of in vivo samples showed a significant extent of HYD in faeces, however, the interaction product was not found in plasma and urine. To verify the results, 5 mg/kg oral dose of HYD standard was given to rats separately, and its presence was studied in all the three matrices. Further, in vitro plasma stability of HYD was also carried out to explain its absence in plasma and urine, which showed ∼55% disappearance of HYD in 2 h.     

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

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

A generic approach for the determination of trace hydrazine in drug substances using in situ derivatization-headspace GC–MS

In situ derivatization-headspace GC–MS methodology has been developed for the determination of hydrazine in drug substance at low ppm levels. This general method uses acetone or acetone-d₆ as the derivatization reagent. The resulting acetone azine or acetone azine-d₁₂ can then be analyzed by headspace GC–MS. The method gives excellent sensitivity with a limit of quantitation (LOQ) as low as 0.1 ppm when the API (active pharmaceutical ingredient) samples are prepared at 10 mg per headspace injection vial. The spike recoveries of hydrazine at the 1 ppm level were between 79% and 117% in various APIs tested. The precisions (%RSD) of six preparations of the hydrazine standards at the concentration of 1 ppm level were typically between 2.7 and 5.6%. A linear range of concentrations from 0.1 to 10 ppm has been demonstrated with R² ≥ 0.999. This general method has been tested in a number of API matrices and successfully applied to the determination of hydrazine in support of API batch releases and process chemistry at GlaxoSmithKline.     

Preparation,characterization and in vitro evaluation of ε-polylysine-loaded polymer blend microparticles for potential pancreatic cancer therapy

Peptide active ingredients show great promise regarding the treatment of various health-endangering diseases. It is reported that L-lysine inhibits the proliferation of several tumour lines in vitro and in vivo. However, proteins and peptide drugs possess certain disadvantages such as in vivo instability and short biological half-life. On the grounds that drug delivery systems can overcome a wide spectrum of bioactive compounds issues, a biopolymeric blend-based microparticulated system capable of delivering ε-polylysine (PLL) was developed. PLL-loaded poly((L)Lactic acid)/poly(D,L-Lactide)-co-poly(ethylene glycol)-based microparticles (PLL-PB-MPs) were prepared and fully characterised exhibiting a narrow size distribution (1.2?±?0.12?µm), high loading efficiency (81%) and improved thermal stability (T_d from 250?°C to 291?°C). The cytotoxicity and antiproliferative effect of PLL-PB-MPs in pancreatic adenocarcinoma cell lines BxPC3 and MIA PaCa-2 were confirmed. Due to their physicochemical and biopharmaceutical properties, PB-MPs constitute a promising carrier to deliver bioactive peptides.     

Identification and characterization of degradation products of irbesartan using LC–MS/TOF,MS, on-line H/D exchange and LC–NMR

Irbesartan was subjected to hydrolytic, oxidative, photolytic and thermal stress, according to ICH guideline Q1A (R2). The drug showed degradation only in acidic, basic and photoacidic conditions, while it was stable to other stress conditions. A total of three degradation products were formed, which were separated on a C-8 column employing a gradient HPLC method. Initially, a complete mass fragmentation pathway of the drug was established with the help of MS/TOF, MSⁿ and H/D exchange studies. Subsequently, the degradation products were subjected to LC–MS/TOF and on-line H/D exchange mass studies to obtain their accurate mass, fragment pattern and number of labile hydrogens. The MS results helped to assign tentative structures to degradation products, which were verified through ¹H and 2D COSY LC–NMR experiments. The products were identified as (2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methanamine, 1-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methylamino)pentylideneamino)cyclopentane carboxylic acid and 2-butyl-3-(tetrazolo[1,5-f]phenanthridin-6-ylmethyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The structures were justified by mechanisms of their formation.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

Development of a LC–MS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of antrodin B and antrodin C in rat plasma. Both target compounds, together with the internal standard (diazepam), were extracted from rat plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Agilent XDB-C₈ column with an isocratic mobile phase consisting of acetonitrile and water (70:30, V/V) at a flow rate of 0.5 mL/min. The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source operating in positive ionization mode. The assay exhibited a linear dynamic range of 47.6–4760 ng/mL for antrodin B and 56.6–5660 ng/mL for antrodin C. The intra- and inter-day precision was less than 5.3% and the accuracy was less than 2.7% for both analytes. The validated method has been applied to the pharmacokinetic study of antrodin B and antrodin C in rats following oral administration of Antrodia camphorata extract.     

Simple and reproducible HPLC–DAD–ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical study on this species roots. Alkaloids in aqueous extracts, the usual form of consumption of this matrix, were studied using HPLC–DAD–ESI-MS/MS, which allowed the identification of 19-S-vindolinine, vindolinine, ajmalicine and an ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer. Quantification of the identified compounds revealed that serpentine and its isomer were predominant (64.7%) over the other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The used procedure revealed to be simple, sensitive and reproducible.     

Non-destructive profiling of volatile organic compounds using HS-SPME/GC–MS and its application for the geographical discrimination of white rice

The authenticity determination of white rice is crucial to prevent deceptive origin labeling and dishonest trading. However, a non-destructive and comprehensive method for rapidly discriminating the geographical origins of white rice between countries is still lacking. In the current study, we developed a volatile organic compound based geographical discrimination method using headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME/GC–MS) to discriminate rice samples from Korea and China. A partial least squares discriminant analysis (PLS-DA) model exhibited a good classification of white rice between Korea and China (accuracy = 0.958, goodness of fit = 0.937, goodness of prediction = 0.831, and permutation test p-value = 0.043). Combining the PLS-DA based feature selection with the differentially expressed features from the unpaired t-test and significance analysis of microarrays, 12 discriminatory biomarkers were found. Among them, hexanal and 1-hexanol have been previously known to be associated with the cultivation environment and storage conditions. Other hydrocarbon biomarkers are novel, and their impact on rice production and storage remains to be elucidated. In conclusion, our findings highlight the ability to rapidly discriminate white rice from Korea and China. The developed method maybe useful for the authenticity and quality control of white rice.     

Development of a linear dual column HPLC–MS/MS method and clinical genetic evaluation for tramadol and its phase I and II metabolites in oral fluid

Tramadol is a centrally acting synthetic opioid analgesic and has received special attention due to its abuse potential and unexpected responses induced by CYP2D6 polymorphism. Oral fluid is an advantageous biofluid for drug analysis due to non-invasive sampling and high correlation of drug concentrations with plasma. However, few studies have been performed on distribution of tramadol and its metabolites in oral fluid. In the present study, a linear dual column HPLC–MS/MS method was developed and fully validated for the simultaneous determination of tramadol and its phase I [O-desmethyltramadol (ODMT), N-desmethyltramadol (NDMT) and N,O-didesmethyltramadol (NODMT)] and II metabolites in oral fluid. Furthermore, the distribution of tramadol and its metabolites, in relation to CYP2D6 genetic variations, in oral fluid was investigated following a clinical study including 23 subjects with CYP2D6*wt/*wt, CYP2D6*10/*10 or CYP2D6*5/*5. The validation results of selectivity, matrix effect, linearity, precision and accuracy were satisfactory. Pharmacokinetic parameters, such as C_ss,max and AUC_0–τ of tramadol, NDMT and NODMT, in the CYP2D6*10/*10 group were significantly higher than those in the CYP2D6*wt/*wt group. Moreover, the ratios of ODMT/tramadol, NDMT/tramadol and NODMT/NDMT correlated well with the CYP2D6 genotypes. We demonstrated that oral fluid is a promising biofluid for pharmacokinetic evaluation in relation to genetic variations.     

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

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

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO₄, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well.     

Identification and characterization of a photolytic degradation product of telmisartan using LC–MS/TOF,LC–MS,LC–NMR and on-line H/D exchange mass studies

Telmisartan, an anti-hypertensive drug, was subjected to stress studies under ICH prescribed conditions of hydrolysis (acidic, neutral and basic), photolysis, oxidation and thermal stress. The drug showed labiality under only photo-acidic condition by forming a single degradation product. HPLC separation of the drug and the degradation product was achieved on C-8 column using gradient method. To characterize the product, a complete mass fragmentation pathway of the drug was initially established. Subsequently, the degradation product peak was subjected to LC–MS/TOF and on-line H/D exchange mass studies. Based on these studies, a tentative structure was assigned to the product as 3-((1,7′-dimethyl-2′-propyl-1H,3′H-2,5′-bibenzo[d]imidazol-3′-yl)methyl)-6H-benzo[c]chromen-6-one, which was verified through ¹H LC–NMR experiments.     

Quality assessment of traditional herbal formula,Hyeonggaeyeongyo-tang through simultaneous determination of twenty marker components by HPLC–PDA and LC–MS/MS

Simultaneous analysis of 20 marker components (gallic acid, cimifugin, geniposide, paeoniflorin, ferulic acid, nodakenin, narirutin, naringin, neohesperidin, arctiin, baicalin, oxypeucedanin hydrate, wogonoside, baicalein, arctigenin, glycyrrhizin, wogonin, pulegone, decursin, and decursinol angelate) for quality assessment of the traditional herbal formula, Hyeonggaeyeongyo-tang (HYT) was carried out by using high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) and liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS/MS). The coefficient of determination showed excellent linearity of more than 0.9999 for all analytes. The recovery of 20 marker components was 93.92 to 102.66% with relative standard deviation (RSD) < 3.00% and RSD value of precision was ≤ 3.44%. The amounts of 20 marker components using HPLC–PDA and LC–MS/MS were determined to be 0.18–14.60 and 0.01–1.76 mg/freeze-dried g, respectively.     

Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC–MS/MS for doping controls

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC–MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45–50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100–1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.