Lung-Cheng Lin’s research while affiliated with ScinoPharm Taiwan and other places

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Publications (13)


Characterizing the D-Amino Acid Position in Peptide Epimers by Using Higher-Energy Collisional Dissociation Tandem Mass Spectrometry: A Case Study of Liraglutide
  • Article
  • Full-text available

January 2024

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78 Reads

International Journal of Molecular Sciences

Yuan-Chih Chen

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Lung-Cheng Lin

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D-amino acid-containing peptides (DAACPs) occur in biological and artificial environments. Since the importance of DAACPs has been recognized, various mass spectrometry-based analytical approaches have been developed. However, the capability of higher-energy collisional dissociation (HCD) fragmentation to characterize DAACP sites has not been evaluated. In this study, we compared the normalized spectra intensity under different conditions of HCD and used liraglutide along with its DAACPs as examples. Our results indicated that the difference in the intensity of y ions between DAACPs and all-L liraglutide could not only distinguish them but also localize the sites of D-amino acids in the DAACPs. Our data demonstrate the potential of using HCD for the site characterization of DAACPs, which may have great impact in biological studies and peptide drug development.

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Assessing the Similarity between Random Copolymer Drug Glatiramer Acetate by Using LC-MS Data Coupling with Hypothesis Testing

October 2019

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75 Reads

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3 Citations

Analytical Chemistry

The full characterization of non-biological complex drugs (NBCDs) is not possible but analytical approaches are of urgent needs to evaluate the similarity between different lots and compare with their follow-up versions. Here, we propose a hypothesis testing-based approach to assess the similarity/difference between random amino acid copolymer drugs using liquid chromatography mass spectrometry (LC-MS) analysis. Two glatiramer acetate (GA) drugs, commercially available Copaxone and in-house synthesized SPT, and a negative control were digested by Lys-C and followed by HILIC-MS analysis. After retention time alignment and feature identification, 1627 features matched to m/z values in an elemental composition database were considered as derived from active drug ingredients. A hypothesis testing approach, sum of squared deviations test, was developed to process high-dimensional data derived from LC-MS spectra. The feasibility of this approach was first demonstrated by testing 5 versus 5 lots of Copaxone and Copaxone versus SPT, which suggested a significant similarity by obtaining the estimated 95th percentile of the distribution of the estimator (ρ (95%)) at 0.0056 (p-value=0.0026) and 0.0026 (p-value<0.0001), respectively. In contrast, the ρ (95%) was 0.036 (p-value=1.00) while comparing Copaxone and the negative control, implying a lack of similarity. We further synthesized 9 stable isotope-labeled peptides to validate the proposed amino acid sequences in the database, demonstrating the correctness in sequence identification. The quantitation variations in our analytical procedures were determined to be 6.8%-7.7%. This approach was found to have a great potential for evaluating the similarity between generic NBCDs and listed reference drugs as well as to monitor the lot-to-lot variation.


Identification of Di-isononyl Phthalate Metabolites for Exposure Marker Discovery Using In Vitro/In Vivo Metabolism and Signal Mining Strategy with LC-MS Data

November 2011

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35 Reads

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15 Citations

Analytical Chemistry

Di-isononyl phthalate esters (DINPs) are endocrine-disrupting chemicals and have replaced di(2-ethylhexyl) phthalate (DEHP) as the major plasticizer for poly(vinyl chloride) (PVC) products in recent years. Exposure marker discovery of DINPs is crucial, because of their high potential for human exposure and toxicity. Here, we propose an alternative approach for tracing signals derived from stable isotope-labeled precursors with varied labeling ratios to efficiently filter probable metabolite signals. The statistical process, which involves a signal mining algorithm with isotope tracing (SMAIT), has effectively filtered 13 probable DINP metabolite signals out of the 8867 peaks in the LC-MS data obtained from incubated stable isotope-labeled precursors with liver enzymes. Seven of the 13 probable metabolite signals were confirmed as DINP structure-related metabolites by preliminary MS/MS analyses. These 7 structure-related metabolite signals were validated as effective DINP exposure markers, using urine samples collected from DINP-administered rats without time-consuming comprehensive structure identification. We propose that the 7 identified possible DINP metabolite signals of m/z 279.1, 293.1, 305.1, 307.1, 321.1, 365.1, and 375.1 are potential markers for DINP exposure and should be investigated further. The integrated approach described here can efficiently, and systematically, filter probable metabolite signals from a complex LC-MS dataset for toxic exposure marker discovery. It is a relatively low-cost/rapid workflow for exposure marker discovery.



Collection of in vivo-like liver cell secretome with alternative sample enrichment method using a hollow fiber bioreactor culture system combined with tangential flow filtration for secretomics analysis

January 2011

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83 Reads

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23 Citations

Analytica Chimica Acta

A hollow fiber bioreactor (HFB) culture system coupled with a tangential flow filtration (TFF) device was used for HepG2 cell secretome analysis. In order to reduce the loss of low-molecular-weight proteins, two new features, the hollow fiber with 0.1 μm pore size and a TFF device with a membrane of 1kDa molecular weight cutoff, were added to the system described previously. The HFB culture system and the conventional dish culture method for secretome collection were compared side by side. It was observed that only a small fraction of cells (<0.01%) were lysed in the HFB culture system, in contrast to the 2.73% in the conventional dish culture. A total of 111 proteins were identified in the collected conditioned medium (CM) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with this improved collection procedure. Many of these proteins reported to be biomarkers for liver-related diseases. About 16% of the identified proteins were smaller than 20kDa, demonstrating that the modified collection system had the ability to reduce the loss of low-molecular-weight proteins, in contrast to our previous collection system. The percentage increase of proteins classified as extracellular space or plasma membrane between the conventional dish culture and the HFB culture system was 40-60%. We believed that in vivo-like culture environments could support liver cells to improve protein secretion than conventional dish cultures. We suggest that the combination of the HFB culture system, TFF device, and LC-MS/MS analysis, would be an efficient procedure for the collection and characterization of in vivo-like cell secretome.



A Statistical Procedure to Selectively Detect Metabolite Signals in LC-MS Data Based on Using Variable Isotope Ratios

October 2009

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24 Reads

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19 Citations

Journal of the American Society for Mass Spectrometry

The tracing of metabolite signals in LC-MS data using stable isotope-labeled compounds has been described in the literature. However, the filtering efficiency and confidence when mining metabolite signals in complex LC-MS datasets can be improved. Here, we propose an additional statistical procedure to increase the compound-derived signal mining efficiency. This method also provides a highly confident approach to screen out metabolite signals because the correlation of varying concentration ratios of native/stable isotope-labeled compounds and their instrumental response ratio is used. An in-house computational program [signal mining algorithm with isotope tracing (SMAIT)] was developed to perform the statistical procedure. To illustrate the SMAIT concept and its effectiveness for mining metabolite signals in LC-MS data, the plasticizer, di-(2-ethylhexyl) phthalate (DEHP), was used as an example. The statistical procedure effectively filtered 15 probable metabolite signals from 3617 peaks in the LC-MS data. These probable metabolite signals were considered structurally related to DEHP. Results obtained here suggest that the statistical procedure could be used to confidently facilitate the detection of probable metabolites from a compound-derived precursor presented in a complex LC-MS dataset.


Association between GST Genetic Polymorphism and Dose-Related Production of Urinary Benzene Metabolite Markers, trans, trans-Muconic Acid and S-Phenylmercapturic Acid

June 2008

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31 Reads

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31 Citations

Cancer Epidemiology Biomarkers & Prevention

The urinary benzene metabolites, trans, trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA), are widely used as benzene exposure biomarkers. The influence of the glutathione S-transferase (GST) genetic polymorphism on the excretion levels of urinary ttMA and/or SPMA has been investigated. The association between dose-related production of urinary benzene metabolites and benzene exposure level was also reported. However, the association between the dose-related productions of urinary benzene metabolites and GST genetic polymorphism was not described in the literature. The purpose of this study was to investigate the association between the GST genetic polymorphism and dose-related production of the two widely used biomarkers, urinary ttMA and SPMA. Seventy male workers in a chemical factory were measured for their benzene exposure levels and provided blood and urine specimens at the end of work-shift. The atmospheric benzene exposure levels of these workers were determined by passive samplers with gas chromatograph mass spectrometer. The urinary ttMA and SPMA levels were quantitated by an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometer. The analyses of GST genotypes, including M(1), T(1), and P(1), were done using PCR. Mean (+/- SD) of benzene exposure levels in participants was 7.2 +/- 15 ppm. The ttMA and SPMA levels in the high benzene exposure group (> or =1 ppm) were higher than those in the low benzene exposure group (<1 ppm; P < 0.001). Among the GST genotypes investigated in this study, the results showed that only the GSTT1 genotype was related to the level and dose-related production of SPMA. Using SPMA for evaluating benzene exposure, the results suggest that the GSTT1 genetic polymorphism, especially in a comparison study between two populations with different GSTT1 genotype frequencies, should be considered. Additionally, the biological exposure index value of SPMA should be set based on the levels of subjects with GSTT1-deficient genotypes for protection of all subjects.


Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry

January 2008

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80 Reads

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15 Citations

Journal of Chromatography B

The levels of estrogens and/or their metabolites play important roles in carcinogenesis, reproductive function, and sexual development during perinatal and adolescence periods. The main purpose of this report was to investigate the applicability of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with electrospray ionization (ESI) and/or atmospheric pressure chemical ionization (APCI) for simultaneous detection of estrone (E1) and its six metabolites. Both positive and negative ionization modes in ESI and APCI were used to evaluate the signal responses of seven target analytes. Among the seven target analytes, five analytes, E1, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, and 2-hydroxyestrone-3-methyl, produced signals with the best signal-to-noise (S/N) ratios in positive APCI-MS/MS mode, while the other two analytes, 2-hydroxyestrone and 4-hydroxyestrone, yielded the best S/N ratios in negative ESI-MS/MS mode. Based on the results of the evaluation, HPLC-APCI-MS/MS with switching between positive and negative modes was recommended for simultaneous detection of E1 and its six metabolites. The proposed analytical scheme was successfully applied in the analysis of cell culture medium of Human liver carcinoma cells treated with varying amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin.


Validation of an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and S-phenylmercapturic acid

January 2006

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76 Reads

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17 Citations

Analytica Chimica Acta

The aim of this study is to validate isotope-dilution electrospray ionization tandem mass spectrometry (ESI–MS–MS) method with a dual-loop cleanup device for simultaneous quantitation of two benzene metabolites, trans, trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA), in human urine. In this study, a pooled blank urine matrix from rural residents was adopted for validation of the analytical method. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of ttMA and SPMA standards spiked into two kinds of urine matrixes over a wide concentration range, 1/32–8-fold biological exposure indices (BEIs) values, showed good linearity (R > 0.9992). The detection limits in pooled urine matrix for ttMA and SPMA were 1.27 and 0.042 μg g−1 creatinine, respectively. For both of ttMA and SPMA, the intra- and inter-day precision values were considered acceptable well below 25% at the various spiked concentrations. The intra- and inter-day apparent recovery values were also considered acceptable (apparent recovery >90%). The ttMA accuracy was estimated by urinary standard reference material (SRM). The accuracy reported in terms of relative error (RE) was 5.0 ± 2.0% (n = 3). The stability of sample storage at 4 or −20 °C were assessed. Urinary ttMA and SPMA were found to be stable for at least 8 weeks when stored at 4 or −20 °C. In addition, urine samples from different benzene exposure groups were collected and measured in this system. Without tedious manual sample preparation procedure, the analytical system was able to quantify simultaneously ttMA and SPMA in less than 20 min.


Citations (11)


... There are analytical methods used to characterize GA or similar polypeptide mixtures described in the literature, for example by amino acid analysis, by size exclusion chromatography, reverse phase liquid chromatography, capillary electrophoresis, peptide mapping, Edman sequencing; however, the proposed method consists of a chromatographic design coupled to a highly selective mass spectrometry detector, which is capable of discerning from the polypeptide mixture the qualitative-quantitative composition of the GA and its sequential amino acids; designed with the objective of being used as a tool for monitoring critical quality attributes (test and mole fraction) of a formulation, to discriminate batches of GA suitable for pharmaceutical use as an injectable solution [3]. In this work, the analytical method to quantify GA as raw material and finished product was established. ...

Reference:

Development and Validation of an LC/MS Method of Glatiramer and Its Sequential Amino Acids, at Pharmaceutical Product
Assessing the Similarity between Random Copolymer Drug Glatiramer Acetate by Using LC-MS Data Coupling with Hypothesis Testing
  • Citing Article
  • October 2019

Analytical Chemistry

... Over the last 10 years, miniaturization has attracted much attention and has driven solvent and sample savings, sample enrichment, rapid sample preparation, and easier automation. Currently, partially or fully automated methods for the analysis of urinary S-PMA have been proposed by using LC-MS/MS systems.10,12,[31][32][33] The GC alternative can be applied only after adding a derivatization step for S-PMA. ...

Validation of an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and S-phenylmercapturic acid
  • Citing Article
  • January 2006

Analytica Chimica Acta

... Dose-response experiments have been employed in toxicant metabolite identification to determine the applicability of metabolite candidates (Hsu et al., 2011). Drug metabolites are expected to induce a dose-based response, and a dose-response experiment can thus be adopted in metabolomics-based data processing for drug metabolite identification. ...

Identification of Di-isononyl Phthalate Metabolites for Exposure Marker Discovery Using In Vitro/In Vivo Metabolism and Signal Mining Strategy with LC-MS Data
  • Citing Article
  • November 2011

Analytical Chemistry

... Lastly, several studies examined maternal phthalate exposure in relation to hormone ratios. For example, in a Taiwanese cohort (n = 155), among people carrying female fetuses, higher third trimester ΣDEHP was associated with reduced free T: E2 [46]. Other studies have reported conflicting results, however, with lower ratios of urinary androgen to estrogen metabolites among participants with higher exposure to ΣDEHP, mono(3-carboxypropyl) phthalate (MCPP), and MEP [44]. ...

Associations between maternal phthalate exposure and cord sex hormones in human infants
  • Citing Article
  • May 2011

Chemosphere

... However, the use of microcarrier-based bioreactor systems often result in large quantities of condition media, which requires extensive downstream processing for EV isolation/purification. A key benefit of hollow-fibre bioreactors is they allow for the retention of the EV product within the culture compartment, resulting is a more concentrated condition media, facilitating downstream processing [173]. A limitation of using these perfusion-based systems, as with most scale out approaches, are the restraints regarding cell number, where additional systems in parallel will be required to scale EV manufacture [172]. ...

Collection of in vivo-like liver cell secretome with alternative sample enrichment method using a hollow fiber bioreactor culture system combined with tangential flow filtration for secretomics analysis
  • Citing Article
  • January 2011

Analytica Chimica Acta

... These approaches can be used to identify not only common metabolites but also uncommon metabolites that cannot be predicted by analyzing known biotransformation reactions. The metabolomics data processing developed for screening drug metabolites include XCMS Online (Gowda et al., 2014), the mass defect filter (MDF) (Zhang et al., 2009), and stable isotope tracing (SIT) (Lin et al., 2010). ...

A Statistical Procedure to Selectively Detect Metabolite Signals in LC-MS Data Based on Using Variable Isotope Ratios
  • Citing Article
  • October 2009

Journal of the American Society for Mass Spectrometry

... Over the last 10 years, miniaturization has attracted much attention and has driven solvent and sample savings, sample enrichment, rapid sample preparation, and easier automation. Currently, partially or fully automated methods for the analysis of urinary S-PMA have been proposed by using LC-MS/MS systems.10,12,[31][32][33] The GC alternative can be applied only after adding a derivatization step for S-PMA. ...

An Online Automatic Sample Cleanup System for the Quantitative Detection of the Benzene Exposure Biomarker S-Phenyimercapturic Acid in Human Urine by Electrospray Ionization Tandem Mass Spectrometry
  • Citing Article
  • May 2002

Journal of Analytical Toxicology

... However, most of these methods determine only one metabolite 18,[20][21][22][23][24][25][26] . Over the past decades, the LC-MS/MS gained widespread use for quantitation of drugs and their metabolites in biological matrices 5,[27][28][29][30][31][32][33][34] . Several methods with isotopic dilutions have been developed on LC-MS/ MS for the determination of t,t-MA and SPMA in urine samples of workers exposed to low levels of benzene 28,35 . ...

Development and validation of an isotope-dilution electrospray ionization tandem mass spectrometry method with an on-line sample clean-up device for the quantitative analysis of the benzene exposure biomarker S-phenylmercapturic acid in human urine
  • Citing Article
  • June 2004

Rapid Communications in Mass Spectrometry

... However, most of these methods determine only one metabolite 18,[20][21][22][23][24][25][26] . Over the past decades, the LC-MS/MS gained widespread use for quantitation of drugs and their metabolites in biological matrices 5,[27][28][29][30][31][32][33][34] . Several methods with isotopic dilutions have been developed on LC-MS/ MS for the determination of t,t-MA and SPMA in urine samples of workers exposed to low levels of benzene 28,35 . ...

An electrospray ionization tandem mass spectrometry based system with an online dual-loop cleanup device for simultaneous quantitation of urinary benzene exposure biomarkerstrans,trans-muconic acid andS-phenylmercapturic acid
  • Citing Article
  • November 2004

Rapid Communications in Mass Spectrometry

... Human and animal waste-borne steroidal hormones are found in the environment at low nanogram per liter concentrations (Hanselman et al., 2003;Khanal et al., 2006); the detection of such concentrations requires advanced analytical techniques (Xu et al., 2006;Hsu et al., 2007;Xu et al., 2008). The estrogens 17b-estradiol (E2) and estrone (E1) are considered highly potent hormone-active compounds; they interfere with the normal operation of the endocrine system and physiologically affect fish and other aquatic vertebrate species at lower concentrations than other endocrinedisrupting compounds. ...

Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry
  • Citing Article
  • January 2008

Journal of Chromatography B