Na Hyun Park

Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Alkaloids with significant therapeutic effects are the main active constituents of Corydalis (C.) species. There are several kinds of alkaloids in C. species associated with diverse alkaloid metabolism in plants, but they are rarely identified. This study aimed to identify diverse alkaloids in C. species by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). METHODS Several types of alkaloids were extracted from C. species using ultrasonication with 70% CH(3)OH, and the extract was partitioned at pH 2 and 12. Separation of alkaloids was achieved by C18 high-performance liquid chromatography (HPLC), and MS/MS analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry. For further confirmation, LC/Fourier transform ion cyclotron resonance (FTICR)-MS was used to obtain accurate mass data and gas chromatography (GC)/MS combined with trimethylsilyl derivatization was applied for identification of the minor alkaloids. RESULTS Thirty-three alkaloids among three different C. species were successfully separated and identified by LC/ESI-MS/MS and LC/FTICR-MS. Structural assignment of individual alkaloids was performed according to MS/MS spectral patterns. For further confirmation, accurate mass data of alkaloids by LC/FTICR-MS were obtained within 5 ppm and the GC/MS data for the trimethylsilyl alkaloids were also obtained. Among 33 alkaloids identified from this study, 13 alkaloids were reported for the first time in the investigated C. species. CONCLUSIONS The LC/ESI-MS/MS technique was effective in obtaining structural information and yielded diagnostic ions for diverse alkaloids. Based on the identified 33 alkaloids, marker compounds were suggested for the three C. species with different geographic origins. This study may also be useful for elucidating unknown alkaloids in herbal medicines.

Profiling analysis of biogenic amines and their acidic metabolites in mouse brain tissue using gas chromatography-tandem mass spectrometry.

The profiling analysis of biogenic amines, including catecholamines and serotonin, and their metabolites in mouse brain tissue provides an important key to understanding their roles in the body and the possibility of simple and accurate diagnosis of neural diseases. A novel approach in the analysis of biogenic amines and their acidic metabolites in brain tissue using gas chromatography-tandem mass spectrometry (GC-MS/MS) is presented. Biogenic amines and their acidic metabolites in brain tissue were effectively separated using a mixed-cation-exchange solid-phase extraction (MCX-SPE) cartridge with a variation in the composition of the SPE elution solvents. A selective derivatization with hexamethyldisilazane (HMDS) and N-methyl-bis-heptafluorobutyramide (MBHFBA) was used to increase the detection sensitivity and to prevent the formation of any side-products. The identification and quantification of the target analytes were performed by gas chromatography triple quadrupole mass spectrometry (GC-MS/MS) using multiple ion reaction monitoring (MRM) mode. The overall recovery yields of the biogenic amines and their metabolites were above 87.5% at 10ng/g and 92.4% at 100ng/g of spiking concentration range. The isotopic-labeled internal standards were used for the precise quantification of bioamines and their metabolites. The calibration curves for the biogenic amines and their metabolites obtained through GC-MS/MS were linear (r(2)>0.995) over the concentration range of 1 (2 or 3)-200ng/mL. The present method was reproducible (relative standard deviation range 0.6-9.3%) and accurate (range 85.4-107.9%), with LLOQs of 0.71-3.69ng/mL. The developed method was successfully applied to the determination and quantification of bioamines and their metabolites in rat brain tissue samples.

Comprehensive impurity profiling and quantification of Sudan III dyes by gas chromatography/mass spectrometry.

A novel analysis strategy was created for comprehensive qualitative and quantitative impurity profiling of the coloring agent Sudan III by gas chromatography/mass spectrometry (GC/MS). The identification of impurities in commercial Sudan III was performed by GC/MS combined with trimethylsilylation (TMS). A total of 24 impurities were identified or tentatively characterized in commercial Sudan III dyes by GC/MS and were mainly classified as phenylazo and naphtholazo analogs. Four new impurities with coplanar structures, suspected of being toxic compounds, were observed in commercial Sudan III dyes. For further identification and sensitive detection of polar impurities, an extract was trimethylsilyl-derivatized to improve the GC chromatographic properties and mass spectrometric detection sensitivity. On the basis of the impurities identified by GC/MS, pathways for the formation of the major impurities during the manufacture of Sudan III were suggested. Four impurities regulated by the EU commission and the US Code of Federal Regulations (CFR) in Sudan III were quantified by GC/MS-scan mode. Method validation was conducted to determine linearity, precision, accuracy, and limit of quantification (LOQ). The linear dynamic range extended from 0.001 to 4.0%, with a correlation coefficient (R(2)) greater than 0.997 for GC/MS. The LOQs of the impurities ranged from 2.73 to 4.39μg/g for GC/MS. Based on the established method, the levels of regulated impurities in five commercial Sudan III dyes manufactured by different chemical companies were successfully determined. This study provides very useful information for the quality control of Sudan III and evaluation of its manufacture.

Metabolite identification of a new tyrosine kinase inhibitor, HM781-36B, and a pharmacokinetic study by liquid chromatography/tandem mass spectrometry

RATIONALE HM781-36B (1-[4-[4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy]-piperidin-1-yl]prop-2-en-1-one hydrochloride) is a new anticancer drug to treat advanced solid tumors in clinical trial. In order to understand the behavior of HM781-36B in vitro and in vivo we validated an analytical method for HM781-36B and its major metabolites in plasma. METHODS In vivo and in vitro metabolism of HM781-36B was studied in dog plasma, urine and feces as well as using human and dog liver microsomes with extraction by ethyl acetate or methyl tert-butyl ether, respectively, and successfully separated by high-performance liquid chromatography diode-array detection mass spectrometry (HPLC-DAD/MS). Ten metabolites were identified by LC/ESI-ion trap mass spectrometry (MS, MS(2) , MS(3) and MRM) and LC/Q-TOF-MS/MS for exact mass measurement. For accurate characterization of the major metabolites, authentic standards (M1, M2, M4, and M10) were synthesized. RESULTS Ten metabolites of HM781-36B in an in vitro mixture were separated and identified by LC/ESI-MS(n) . The MS/MS spectral patterns of the parent drug and metabolites exhibited two characteristic ions (A- and B-type ions) attributed to the cleavage of the ether bond between the piperidine ring and the quinazoline ring, providing important information on the site of chemical conversion during the metabolism. Six hydroxylated derivatives including dehalogenation and demethylation, two N-oxide forms, a demethylated form and de-acryloylpiperideine metabolites were observed. CONCLUSIONS The LC/ESI-ion trap MS(n) technique was effective in obtaining structural information and yielded diagnostic ions for the identification of diverse metabolites. The multiple metabolic pathways of HM781-36B were suggested in in vitro and in vivo samples and the dihydroxylation (M1) and demethylation (M2) appeared to be the major metabolites.

Metabolic profiling of tyrosine, tryptophan, and glutamate in human urine using gas chromatography–tandem mass spectrometry combined with single SPE cleanup

The tyrosine, tryptophan, and glutamate metabolic pathways play key roles on pathological state of neuronal functions and the change of their levels in biological systems reflects the progress degree of neuronal diseases. Comprehensive profiling of these metabolites is important to find new biomarkers for diagnosis or prognosis of various neuronal diseases. However, the overall profiling analysis of various neurochemicals in biological sample is confronted with several limitations due to their low concentration and physicochemical properties and the coexistence of matrices. We developed an efficient and feasible method using gas chromatography-tandem mass spectrometry (GC-MS/MS). Wide-bore mixed cation exchange (MCX) SPE process enables a rapid and effective cleanup of 20 neurochemicals even including acidic and basic neurochemicals in a single SPE cartridge by using different composition of eluents. Selective derivatization of various types of metabolites was applied to achieve highly chromatographic separation and sensitive mass detection. Appropriate selection of precursor and product transition ions used in multiple reaction-monitoring (MRM) mode based on the MS/MS fragmentations of the derivatized neurochemicals could be significantly minimized the matrix effects and enhanced the reliability of quantification results. The developed method was validated in terms of linearity, limits of detection, precision, accuracy, and matrix effects. The intra- and inter-assay analytical variations were less than 10%. The overall linearity for all of the targets was excellent (R2≥0.996). The detection limits ranged between 0.38 and 8.13ng/mL for the acidic neurochemicals and between 0.02 and 11.1ng/mL for the basic neurochemicals. The developed protocol will be expected to be a promising tool for the understanding of the pathological state and diagnosis of various neuronal diseases.

Comprehensive chemical profiling of Pinellia species tuber and processed Pinellia tuber by gas chromatography–mass spectrometry and liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

A comprehensive profiling method was established for the determination of various chemicals in Pinellia (P.) ternata and pedatisecta species. The profiling method comprises a fast ultrasonic extraction with various solvents, followed by GC-MS and LC-APCI-MS analysis. A total of 73 polar components as trimethylsilyl (TMS) derivatives were detected in methanol extract by GC-MS. The main components of the P. species were profiled as several kinds of fatty acids, amino acids, nucleic acids, carbohydrates, and phenolic compounds. The hexane extract was analyzed by LC-APCI-MS for the lipid profiling. A total of 35 lipid constituents [fatty acids and their esters, mono-, di-, and tri-acylglycerols] and four phytosterols were observed and tentatively characterized by LC-APCI-MS/MS. Among the phytochemicals detected in the hexane extract, triacylglycerols (TAGs) as the major component were identified by LC-APCI-MS and MS/MS. Based on the identified components, a significant difference in the chemical compositions of P. species tuber and processed P. ternata was found that the complete disappearance of TAGs and a considerable decrement of sucrose were observed in processed P. ternata. Furthermore, the degradation mechanism for TAGs in the presence of alum solution is suggested to occur during the processing P. ternata. Malic acid was found to be a characteristic compound for the classification of P. ternata and pedatisecta with different geographic origins. Based on the validated GC/MS method, twenty-four P. ternata, processed P. ternata and P. pedatisecta samples were profiled to measure the overall abundance of specific groups of compound and to identify diagnostic compounds. In addition, principal component analysis (PCA) on the GC/MS profiling data revealed a clear classification of P. species samples. In this study, the full chemical complement was for the first time reported for quality evaluation of P. species. The method can be usefully applied for phytochemical analysis of related herbal medicines.

Profiling of a wide range of neurochemicals in human urine by very-high-performance liquid chromatography-tandem mass spectrometry combined with in situ selective derivatization

Development of a reliable analytical method of neurochemicals in biological fluids is important to discover potential biomarkers for the diagnosis, treatment and prognosis of neurological disorders. However, neurochemical profiling of biological samples is challenging because of highly different polarities between basic and acidic neurochemicals, low physiological levels, and high matrix interference in biological samples. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method combined with in situ selective derivatization for comprehensive profiling of 20 neurochemicals in urine was developed for a wide range of neurochemicals. In situ selective derivatization greatly improved the peak capacity on a reversed-phase C18 column and sensitive mass detection in LC-ESI-MS/MS-positive ion mode due to reduction of the distinct physicochemical properties between acidic and basic neurochemicals. The MS/MS spectra of neurochemicals exhibited specific ions, such as losses of amine, methanol, or methyl formate molecules from protonated molecules, enabling selection of appropriate multiple reaction monitoring (MRM) ions for selective and sensitive detection. The developed method was validated in terms of linearity, limit of detection (LOD) and limit of quantification (LOQ), precision, accuracy, and recovery. The correlation coefficients (R2) of calibration curves were above 0.9961. The ranges of LODs and LOQs were 0.1-3.6ng/mL and 0.3-12.0ng/mL, respectively. The overall precision and accuracy were 0.52-16.74% and 82.26-118.17%, respectively. The method was successfully applied to simultaneously profile the metabolic pathways of tyrosine, tryptophan, and glutamate in Parkinsons disease patient urine (PD, n=21) and control urine (n=10). Significant differences (P≤0.01) between two groups in the activity of phenylethanolamine N-methyltransferase (PNMT) and alcohol dehydrogenase (ADH) were observed. In conclusion, this method provides reliable quantification of a wide range of neurochemicals in human urine and would be helpful for finding biomarkers related to specific neuronal diseases.

An in situ extraction and derivatization method for rapid analysis of short-chain fatty acids in rat fecal samples by gas chromatography tandem mass spectrometry

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and changes in their levels in a biological system reflect the progress of various diseases. However, analysis of SCFAs in biological samples is limited due to their easy loss during sample workup, their low concentrations, and the coexistence of matrices. In this study, we developed a feasible method for the analysis of SCFAs in rat fecal samples using gas chromatography-tandem mass spectrometry (GC-MS/MS) combined with an in situ extraction and derivatization method. More specifically, in situ extraction and derivatization were applied for the determination of SCFAs using tetrabutylammonium hydrogen sulfate (TBAHS) and pentafluorobenzyl bromide (PFBBr). TBAHS concentration and pH values, as well as the temperature and time of the derivatization reaction were effectively optimized. Moreover, the proposed process reduced the analysis time and limited the loss of SCFAs during the sample workup. It also improved their peak shapes and resulted in longer GC retentions and high sensitivity in MS detection. Use of GC-MS/MS could offer high sensitivity and selectivity for SCFA-PFB derivatives in complicated matrix samples by selecting a multiple-ion reaction monitoring (MRM) transition ion at m/z 181 that corresponds to the [PFB]+ ion. The established method was also validated in terms of linearity, limits of detection (LODs) and quantification (LOQs), and matrix effects. It was proved to be linear (r2 > 0.997), with lower LODs of 5–24 ng mL−1, and lower LOQs of 0.05–0.1 μg mL−1 for all SCFAs. The proposed method will be useful for the determination of SCFAs in rat fecal samples and for the clinical diagnosis of various diseases.