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2013 Volume 31 Issue 10
2013, 31(10): 925-926
doi: 10.3724/SP.J.1123.2013.09025
Abstract:
2013, 31(10): 927-933
doi: 10.3724/SP.J.1123.2013.04041
Abstract:
Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine>N-terminal amino acid>aspartic acid>glutamic acid>histidine>asparagine>lysine>tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.
Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine>N-terminal amino acid>aspartic acid>glutamic acid>histidine>asparagine>lysine>tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.
2013, 31(10): 934-938
doi: 10.3724/SP.J.1123.2013.01048
Abstract:
An analytical method was developed for the determination of total residues of ribavirin and its phosphorylated metabolites in chicken and its products by hydrophilic interaction chromatography-tandem mass spectrometry. The samples were extracted with acetonitrile containing 1% (v/v) acetic acid under ultrasonic condition and then enzymatically hydrolysed with acid phosphatase at 37 ℃. After liposoluble substances being removed with hexane, C18 and PSA dispersion solid phase extraction (DSPE) was introduced to cleanup procedures. The separation was performed on an ultra-performance hydrophilic interaction chromatographic (HILIC) amide column under a gradient elution using acetonitrile and 0.1% formic acid. The analytes were detected in the positive electrospray ionization and multi-reaction monitoring (MRM) mode. In the range of 1-200 μg/kg, the correlation coefficient was greater than 0.999. The limit of detection (LOD, S/N≥3) was 1 μg/kg and the limit of quantification (LOQ, S/N≥10) was 5 μg/kg. The recoveries of ribavirin spiked at three levels ranged from 67.8% to 112.7% with the relative standard deviations (RSDs) of 6.1%-13.6%. The results of the determination of ribavirin in various real samples showed that the method is simple, rapid, accurate and suitable for the determination of total residues of ribavirin and its metabolites in chicken and its products.
An analytical method was developed for the determination of total residues of ribavirin and its phosphorylated metabolites in chicken and its products by hydrophilic interaction chromatography-tandem mass spectrometry. The samples were extracted with acetonitrile containing 1% (v/v) acetic acid under ultrasonic condition and then enzymatically hydrolysed with acid phosphatase at 37 ℃. After liposoluble substances being removed with hexane, C18 and PSA dispersion solid phase extraction (DSPE) was introduced to cleanup procedures. The separation was performed on an ultra-performance hydrophilic interaction chromatographic (HILIC) amide column under a gradient elution using acetonitrile and 0.1% formic acid. The analytes were detected in the positive electrospray ionization and multi-reaction monitoring (MRM) mode. In the range of 1-200 μg/kg, the correlation coefficient was greater than 0.999. The limit of detection (LOD, S/N≥3) was 1 μg/kg and the limit of quantification (LOQ, S/N≥10) was 5 μg/kg. The recoveries of ribavirin spiked at three levels ranged from 67.8% to 112.7% with the relative standard deviations (RSDs) of 6.1%-13.6%. The results of the determination of ribavirin in various real samples showed that the method is simple, rapid, accurate and suitable for the determination of total residues of ribavirin and its metabolites in chicken and its products.
2013, 31(10): 939-945
doi: 10.3724/SP.J.1123.2013.03042
Abstract:
An ultra high performance liquid chromatography-tandem mass spectrometric method (UHPLC-MS/MS) was developed for the simultaneous determination of three natural forms of azaspiracids (AZA-1, AZA-2 and AZA-3) in edible shellfishes such as mussels, oysters, clams and scallops. The samples were homogeneously extracted with acetonitrile-water (85:15, v/v). The resultant supernatants were purified with QuEChERS method and filtrated by 0.2 μm microporous filters. The separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm) with the gradient elution using acetonitrile/water (containing 5 mmol/L ammonium acetate and 0.1% formic acid) as mobile phases. The three azaspiracids were detected using positive electrospray ionization (ESI+) followed with multiple reaction monitoring (MRM), and quantified by external standard calibration method. The calibration curves showed good linearity in the range of 1-100 μg/kg with the correlation coefficients (r2) greater than 0.995. The limits of quantification (LOQ, S/N=10) were 1.0 μg/kg for all the three AZAs. The average recoveries of azaspiracids spiked in the matrix at the levels of 10, 20 and 50 μg/kg ranged from 71% to 108%. The relative standard deviations (RSDs) of inter-day and intra-day determinations were less than 10%(n=6). The samples from several areas within and outside of China were screened, and some of the samples showed positive for azaspiracids. The developed method is easy to operate, very reproducible, sensitive, and efficient. It can be applied to the determination of the three forms of AZAs in the edible shellfishes as well shellfish products.
An ultra high performance liquid chromatography-tandem mass spectrometric method (UHPLC-MS/MS) was developed for the simultaneous determination of three natural forms of azaspiracids (AZA-1, AZA-2 and AZA-3) in edible shellfishes such as mussels, oysters, clams and scallops. The samples were homogeneously extracted with acetonitrile-water (85:15, v/v). The resultant supernatants were purified with QuEChERS method and filtrated by 0.2 μm microporous filters. The separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm) with the gradient elution using acetonitrile/water (containing 5 mmol/L ammonium acetate and 0.1% formic acid) as mobile phases. The three azaspiracids were detected using positive electrospray ionization (ESI+) followed with multiple reaction monitoring (MRM), and quantified by external standard calibration method. The calibration curves showed good linearity in the range of 1-100 μg/kg with the correlation coefficients (r2) greater than 0.995. The limits of quantification (LOQ, S/N=10) were 1.0 μg/kg for all the three AZAs. The average recoveries of azaspiracids spiked in the matrix at the levels of 10, 20 and 50 μg/kg ranged from 71% to 108%. The relative standard deviations (RSDs) of inter-day and intra-day determinations were less than 10%(n=6). The samples from several areas within and outside of China were screened, and some of the samples showed positive for azaspiracids. The developed method is easy to operate, very reproducible, sensitive, and efficient. It can be applied to the determination of the three forms of AZAs in the edible shellfishes as well shellfish products.
2013, 31(10): 946-953
doi: 10.3724/SP.J.1123.2013.03016
Abstract:
A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the determination of eight compounds in milk and milk powder. They are four penicillins (penicillin G, penicillin V, amoxicillin and ampicillin) and four major β-lactamase enzymatic metabolites of them (penilloic acid G, penilloic acid V, amoxiilloic acid and ampilloic acid). The compounds were extracted from the samples with acetonitrile and water, cleaned-up by HLB solid-phase extraction cartridges, and then detected by HPLC-MS/MS and quantified by external standard method. The linearities were satisfactory with the correlation coefficients >0.99 at the mass concentrations ranging from 4 μg/L to 200 μg/L for penicillins and from 10 μg/L to 500 μg/L for enzymatic metabolites. The limits of detection and the limits of quantification were 5-50 μg/kg (S/N≥3) and 8-100 μg/kg (S/N≥10), respectively. The average recoveries of the eight compounds were 83.48%-96.97% in milk and 82.70%-95.14% in milk powder. The relative standard deviations (RSDs) in milk and milk powder were 3.86%-10.87% and 3.02%-9.81%, respectively. In conclusion, the established method is convenient, accurate and sensitive so that it can be applied to the determination of penicillin residues and enzymatic metabolites in milk and milk powder.
A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the determination of eight compounds in milk and milk powder. They are four penicillins (penicillin G, penicillin V, amoxicillin and ampicillin) and four major β-lactamase enzymatic metabolites of them (penilloic acid G, penilloic acid V, amoxiilloic acid and ampilloic acid). The compounds were extracted from the samples with acetonitrile and water, cleaned-up by HLB solid-phase extraction cartridges, and then detected by HPLC-MS/MS and quantified by external standard method. The linearities were satisfactory with the correlation coefficients >0.99 at the mass concentrations ranging from 4 μg/L to 200 μg/L for penicillins and from 10 μg/L to 500 μg/L for enzymatic metabolites. The limits of detection and the limits of quantification were 5-50 μg/kg (S/N≥3) and 8-100 μg/kg (S/N≥10), respectively. The average recoveries of the eight compounds were 83.48%-96.97% in milk and 82.70%-95.14% in milk powder. The relative standard deviations (RSDs) in milk and milk powder were 3.86%-10.87% and 3.02%-9.81%, respectively. In conclusion, the established method is convenient, accurate and sensitive so that it can be applied to the determination of penicillin residues and enzymatic metabolites in milk and milk powder.
2013, 31(10): 954-960
doi: 10.3724/SP.J.1123.2013.04028
Abstract:
A method was developed for the simultaneous determination of mepanipyrim, silthiofam, boscalid, fluopicolide, mandipropamid, cyflufenamid in vegetables and fruits by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes were extracted from the samples by acetonitrile and purified by Florisil SPE. The six novel amide fungicides were separated on a Poroshell 120 EC-C18 column with the mobile phases of water and acetonitrile, and finally detected by MS/MS with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, quantified by external standard method. Under the optimal analytical conditions, the correlation coefficients (r) of the six novel amide fungicides were not lower than 0.9990 in the concentration range from 0.5 to 100 μg/L. The limits of detection (S/N≥3) of the method were 0.15 μg/kg for boscalid, silthiofam, mandipropamid, cyflufenamid, 0.10 μg/kg for mepanipyrim and 0.17 μg/kg for fluopicolide. The recovery tests were performed for the 7 types of vegetables and the 3 types of fruits at the spiked levels of 0.5, 5 and 50 μg/kg, and the recoveries of the six analytes ranged from 65% to 124% with the relative standard deviations (RSDs, n=5) of 1%-18%. The matrix effects in vegetables and fruits of the six amide fungicides were significantly reduced by the purification of Florisil SPE compared with the modified QuEChERS. The method is easy, fast, sensitive and accurate, and can meet the requirements of the determination of the six amide fungicide residues in vegetables and fruits.
A method was developed for the simultaneous determination of mepanipyrim, silthiofam, boscalid, fluopicolide, mandipropamid, cyflufenamid in vegetables and fruits by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes were extracted from the samples by acetonitrile and purified by Florisil SPE. The six novel amide fungicides were separated on a Poroshell 120 EC-C18 column with the mobile phases of water and acetonitrile, and finally detected by MS/MS with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, quantified by external standard method. Under the optimal analytical conditions, the correlation coefficients (r) of the six novel amide fungicides were not lower than 0.9990 in the concentration range from 0.5 to 100 μg/L. The limits of detection (S/N≥3) of the method were 0.15 μg/kg for boscalid, silthiofam, mandipropamid, cyflufenamid, 0.10 μg/kg for mepanipyrim and 0.17 μg/kg for fluopicolide. The recovery tests were performed for the 7 types of vegetables and the 3 types of fruits at the spiked levels of 0.5, 5 and 50 μg/kg, and the recoveries of the six analytes ranged from 65% to 124% with the relative standard deviations (RSDs, n=5) of 1%-18%. The matrix effects in vegetables and fruits of the six amide fungicides were significantly reduced by the purification of Florisil SPE compared with the modified QuEChERS. The method is easy, fast, sensitive and accurate, and can meet the requirements of the determination of the six amide fungicide residues in vegetables and fruits.
2013, 31(10): 961-968
doi: 10.3724/SP.J.1123.2013.04037
Abstract:
A method of high performance liquid chromatography-linear ion trap/Orbitrap high resolution mass spectrometry (HPLC-LTQ/Orbitrap MS) was used to screen and confirm carcinogenic dyes in textiles. The analytes were extracted from textile samples with pyridine/water (1/1, v/v) in a water bath under controlled conditions (95 ℃, 150 r/min), and then filtered with a 0.22 μm polytetrafluoroetylene (PTFE) membrane. The eluates were separated on a CAPCELL PAK C18 column (100 mm×2.0 mm, 5 μm) using gradient elution with acetonitrile/5 mmol/L ammonium acetate aqueous solution containing 0.01% formic acid (in positive mode) and acetonitrile/5 mmol/L ammonium acetate aqueous solution (in negative mode), and finally detected by HPLC-LTQ/Orbitrap MS in ESI modes. Full scan experiments were performed over the range of m/z 200-800. The screening and quantitative analysis were carried out by the accurate mass of quasi-molecular ion and the peak area in extracted chromatogram with accurate mass, respectively. The confirmatory analysis for target compounds was performed with the retention time and qualitative fragments obtained by data-dependent scan mode. Under the optimal conditions, nine carcinogenic dyes were routinely detected with mass accuracy below 5×10-6 (5 ppm), and good linearities were provided in their respective linear ranges with correlation coefficients higher than 0.99. The limits of detection were in the range of 0.125-25 mg/kg. The average recoveries at three spiked levels were in the range of 62.13%-116.28% with the relative standard deviations (RSDs) lower than 15%. The proposed method was applied to screen and confirm the nine carcinogenic dyes in textile samples. It is convenient and reliable.
A method of high performance liquid chromatography-linear ion trap/Orbitrap high resolution mass spectrometry (HPLC-LTQ/Orbitrap MS) was used to screen and confirm carcinogenic dyes in textiles. The analytes were extracted from textile samples with pyridine/water (1/1, v/v) in a water bath under controlled conditions (95 ℃, 150 r/min), and then filtered with a 0.22 μm polytetrafluoroetylene (PTFE) membrane. The eluates were separated on a CAPCELL PAK C18 column (100 mm×2.0 mm, 5 μm) using gradient elution with acetonitrile/5 mmol/L ammonium acetate aqueous solution containing 0.01% formic acid (in positive mode) and acetonitrile/5 mmol/L ammonium acetate aqueous solution (in negative mode), and finally detected by HPLC-LTQ/Orbitrap MS in ESI modes. Full scan experiments were performed over the range of m/z 200-800. The screening and quantitative analysis were carried out by the accurate mass of quasi-molecular ion and the peak area in extracted chromatogram with accurate mass, respectively. The confirmatory analysis for target compounds was performed with the retention time and qualitative fragments obtained by data-dependent scan mode. Under the optimal conditions, nine carcinogenic dyes were routinely detected with mass accuracy below 5×10-6 (5 ppm), and good linearities were provided in their respective linear ranges with correlation coefficients higher than 0.99. The limits of detection were in the range of 0.125-25 mg/kg. The average recoveries at three spiked levels were in the range of 62.13%-116.28% with the relative standard deviations (RSDs) lower than 15%. The proposed method was applied to screen and confirm the nine carcinogenic dyes in textile samples. It is convenient and reliable.
2013, 31(10): 969-973
doi: 10.3724/SP.J.1123.2013.03010
Abstract:
A fast analytical method was developed for the determination of N-methyl,propyl-morpholinium cation (MPMo) without UV absorption group by ion-pair chromatography-indirect UV detection. Chromatographic separation was performed on a reversed-phase silica-based monolithic column using background UV absorption reagent aqueous solution-ion-pair reagent aqueous solution-organic solvent as the mobile phase. The effects of the background UV absorption reagent, detection wavelength, ion-pair reagent, organic solvent, column temperature and flow rate on the determination of MPMo were investigated. It was found that the morpholinium cation could be determined well with (0.5 mmol/L 4-aminophenol hydrochloride-0.1 mmol/L 1-heptanesulfonic sodium) aqueous solution-methanol (9:1, v/v) as mobile phase at the UV detection wavelength of 230 nm, the column temperature of 30 ℃ and the flow rate of 1.0 mL/min. Under these conditions, the retention time of MPMo was 2.966 min. The limit of detection for MPMo was 0.07 mg/L (S/N=3). The relative standard deviations (n=5) for the peak area and retention time were 2.1% and 0.02%, respectively. The method was successfully applied to the determination of morpholinium ionic liquid synthesized by a chemistry laboratory. Recovery of MPMo after spiking was 98.8%. The results showed that this method is simple and rapid.
A fast analytical method was developed for the determination of N-methyl,propyl-morpholinium cation (MPMo) without UV absorption group by ion-pair chromatography-indirect UV detection. Chromatographic separation was performed on a reversed-phase silica-based monolithic column using background UV absorption reagent aqueous solution-ion-pair reagent aqueous solution-organic solvent as the mobile phase. The effects of the background UV absorption reagent, detection wavelength, ion-pair reagent, organic solvent, column temperature and flow rate on the determination of MPMo were investigated. It was found that the morpholinium cation could be determined well with (0.5 mmol/L 4-aminophenol hydrochloride-0.1 mmol/L 1-heptanesulfonic sodium) aqueous solution-methanol (9:1, v/v) as mobile phase at the UV detection wavelength of 230 nm, the column temperature of 30 ℃ and the flow rate of 1.0 mL/min. Under these conditions, the retention time of MPMo was 2.966 min. The limit of detection for MPMo was 0.07 mg/L (S/N=3). The relative standard deviations (n=5) for the peak area and retention time were 2.1% and 0.02%, respectively. The method was successfully applied to the determination of morpholinium ionic liquid synthesized by a chemistry laboratory. Recovery of MPMo after spiking was 98.8%. The results showed that this method is simple and rapid.
2013, 31(10): 974-979
doi: 10.3724/SP.J.1123.2013.03026
Abstract:
A mammary gland bioreactor can efficiently express human recombinant monoclonal antibody. However, the target products are similar to the bovine antibody in the raw emulsion material in properties and structures. Thus it is difficult to achieve effective separation of the target products. In this work, the species differences between bovine antibody and recombinant human antibody were analyzed and a new separation strategy was raised based on it. We employed two kinds of affinity chromatography to separate these two antibodies from each other and studied the effect of elution mode upon separation. The results demonstrated that Protein A affinity chromatography could get hybrid antibodies using gradient elution mode, but hardly separate the recombinant human antibody and bovine antibody from each other. In contrast, the combination of Protein A affinity chromatography and displacement chromatography could separate the hybrid antibodies effectively and finally give recombinant human IgG (rHGG) product with the purity of 95%and the yield of more than 95%. Immuno-affinity chromatography could also effectively purify recombinant monoclonal antibodies and owned better generality, which could be used in purification of recombinant antibody expressed by any animal mammary gland.
A mammary gland bioreactor can efficiently express human recombinant monoclonal antibody. However, the target products are similar to the bovine antibody in the raw emulsion material in properties and structures. Thus it is difficult to achieve effective separation of the target products. In this work, the species differences between bovine antibody and recombinant human antibody were analyzed and a new separation strategy was raised based on it. We employed two kinds of affinity chromatography to separate these two antibodies from each other and studied the effect of elution mode upon separation. The results demonstrated that Protein A affinity chromatography could get hybrid antibodies using gradient elution mode, but hardly separate the recombinant human antibody and bovine antibody from each other. In contrast, the combination of Protein A affinity chromatography and displacement chromatography could separate the hybrid antibodies effectively and finally give recombinant human IgG (rHGG) product with the purity of 95%and the yield of more than 95%. Immuno-affinity chromatography could also effectively purify recombinant monoclonal antibodies and owned better generality, which could be used in purification of recombinant antibody expressed by any animal mammary gland.
2013, 31(10): 980-988
doi: 10.3724/SP.J.1123.2013.04043
Abstract:
Matrix effect is an important interfering factor in LC-MS quantitative analysis. In this paper, matrix effects and retention efficiencies of 33 veterinary drugs spiked in river water were studied on hydrophilic-lipophilic balance (HLB) cartridges of 3 brands (Waters, Supelco, and CNW), using LC-MS/MS for detection and reverse osmosis (RO) water as the control under 500-fold concentration. In RO water, only the exogenous matrix effects were observed on three brands of HLB cartridges. Most quinolones and tetracyclines showed positive matrix effects. Estrogens showed negative matrix effects on two brands of HLB cartridges. Sulfonamides were not obviously affected by matrix effects. Chloramphenicols showed negative matrix effects on one brand of HLB cartridge. In river water, matrix effects were different from those of the RO water due to the combined exogenous and endogenous interfering substances. Sulfonamides showed slight matrix effects as those in RO water. Most quinolones and tetracyclines showed positive matrix effects. Chloramphenicols and estrogens showed negative matrix effects. Compared to the external standard method, matrix matched calibration method effectively overcame the matrix effects with better quantitative results. The recoveries of 33 target veterinary drugs spiked in river water at 50 ng/L and 200 ng/L levels were in the ranges of 40.3%-146.0% (Waters), 37.8%-104.2% (Supelco), and 52.9%-150.1% (CNW) with RSDs (n=4) of 0.2%-14.6%. The results indicated that there was no significant difference in the retention efficiency between the 3 HLB cartridges with the matrix matched calibration method. This study provided supporting data for the HLB cartridge selection in multi-residual determination of the veterinary drugs in river water samples.
Matrix effect is an important interfering factor in LC-MS quantitative analysis. In this paper, matrix effects and retention efficiencies of 33 veterinary drugs spiked in river water were studied on hydrophilic-lipophilic balance (HLB) cartridges of 3 brands (Waters, Supelco, and CNW), using LC-MS/MS for detection and reverse osmosis (RO) water as the control under 500-fold concentration. In RO water, only the exogenous matrix effects were observed on three brands of HLB cartridges. Most quinolones and tetracyclines showed positive matrix effects. Estrogens showed negative matrix effects on two brands of HLB cartridges. Sulfonamides were not obviously affected by matrix effects. Chloramphenicols showed negative matrix effects on one brand of HLB cartridge. In river water, matrix effects were different from those of the RO water due to the combined exogenous and endogenous interfering substances. Sulfonamides showed slight matrix effects as those in RO water. Most quinolones and tetracyclines showed positive matrix effects. Chloramphenicols and estrogens showed negative matrix effects. Compared to the external standard method, matrix matched calibration method effectively overcame the matrix effects with better quantitative results. The recoveries of 33 target veterinary drugs spiked in river water at 50 ng/L and 200 ng/L levels were in the ranges of 40.3%-146.0% (Waters), 37.8%-104.2% (Supelco), and 52.9%-150.1% (CNW) with RSDs (n=4) of 0.2%-14.6%. The results indicated that there was no significant difference in the retention efficiency between the 3 HLB cartridges with the matrix matched calibration method. This study provided supporting data for the HLB cartridge selection in multi-residual determination of the veterinary drugs in river water samples.
2013, 31(10): 989-994
doi: 10.3724/SP.J.1123.2013.04005
Abstract:
A method of gas chromatography-mass spectrometry (GC-MS) with liquid-liquid extraction has been developed for the simultaneous determination of 23 ester compounds including acetate esters, acrylic esters, metacrylic acid esters and phthalate acid esters in cigarette water-borne adhesives. After dispersed in water, the sample was extracted by n-hexane solution containing phenyl ethyl propionate as internal standard substance. Then, the solution was centrifuged and filtrated through a 0.45 μm organic membrane filter. Finally, the solution was separated on a DB-WAXETR column (60 m×0.25 mm×0.25 μm), and detected with MS in selected ion monitoring mode, and quantified by internal standard method. The results showed a good linear correlation in the range of 0.4-50.0 mg/L. The recoveries of the ester compounds spiked in the sample were 81.8%-109.1%, and the relative standard deviations (RSDs, n=5) were less than 4%. The limits of detection (LODs) and limits of quantification (LOQs) were in the ranges of 0.02-0.76 mg/kg and 0.04-2.52 mg/kg, respectively. The method is simple, time-saving, and has high sensitivity and good reproducibility. It can be applied to the determination of the 23 ester compounds in cigarette water-borne adhesives.
A method of gas chromatography-mass spectrometry (GC-MS) with liquid-liquid extraction has been developed for the simultaneous determination of 23 ester compounds including acetate esters, acrylic esters, metacrylic acid esters and phthalate acid esters in cigarette water-borne adhesives. After dispersed in water, the sample was extracted by n-hexane solution containing phenyl ethyl propionate as internal standard substance. Then, the solution was centrifuged and filtrated through a 0.45 μm organic membrane filter. Finally, the solution was separated on a DB-WAXETR column (60 m×0.25 mm×0.25 μm), and detected with MS in selected ion monitoring mode, and quantified by internal standard method. The results showed a good linear correlation in the range of 0.4-50.0 mg/L. The recoveries of the ester compounds spiked in the sample were 81.8%-109.1%, and the relative standard deviations (RSDs, n=5) were less than 4%. The limits of detection (LODs) and limits of quantification (LOQs) were in the ranges of 0.02-0.76 mg/kg and 0.04-2.52 mg/kg, respectively. The method is simple, time-saving, and has high sensitivity and good reproducibility. It can be applied to the determination of the 23 ester compounds in cigarette water-borne adhesives.
2013, 31(10): 995-1000
doi: 10.3724/SP.J.1123.2013.03055
Abstract:
A novel type of glycopolymer brushes grafted open-tubular capillary column (OTCC) was developed for the polar compound separation. Briefly, the glycerol methacrylate (GMA) polymer brushes were grafted on the inner wall of OTCC by the surface initiated atom transfer radical polymerization (SI-ATRP). Next, glucose was coupled to the side chains of the GMA polymer brushes to obtain the hydrophilic stationary phase (PGMA-N-Glucose). The optimized SI-ATRP reaction conditions were GMA/CuCl/CuCl2/cyclohexanol system grafting at 25 ℃ for 1 h. After the glucose coupling, the column back pressure was about 3585 kPa. The structure of the glycopolymer brushes on the inner surface of OTCC was characterized by scanning electron microscopy (SEM). The glycopolymer grafting resulted in the formation of three-dimensional wave-like polymer structure on the inner surface of OTCC and largely increased the interior surface area. Therefore, improved column efficiency and loading capacity can be achieved. Under the optimized conditions, the electro osmotic flow (EOF) strength of the glycopolymer brushes grafted OTCC was obviously less than that of the bare column when the pH value of the mobile phase ranging from 3 to 11. Using the glycopolymer brushes grafted OTCC, the baseline separations of polar molecules and proteins were obtained without peak tailing. The future work will focus on the further development of the glycopolymer brushes for the highly polar compound separation, such as glycans and glycoproteins.
A novel type of glycopolymer brushes grafted open-tubular capillary column (OTCC) was developed for the polar compound separation. Briefly, the glycerol methacrylate (GMA) polymer brushes were grafted on the inner wall of OTCC by the surface initiated atom transfer radical polymerization (SI-ATRP). Next, glucose was coupled to the side chains of the GMA polymer brushes to obtain the hydrophilic stationary phase (PGMA-N-Glucose). The optimized SI-ATRP reaction conditions were GMA/CuCl/CuCl2/cyclohexanol system grafting at 25 ℃ for 1 h. After the glucose coupling, the column back pressure was about 3585 kPa. The structure of the glycopolymer brushes on the inner surface of OTCC was characterized by scanning electron microscopy (SEM). The glycopolymer grafting resulted in the formation of three-dimensional wave-like polymer structure on the inner surface of OTCC and largely increased the interior surface area. Therefore, improved column efficiency and loading capacity can be achieved. Under the optimized conditions, the electro osmotic flow (EOF) strength of the glycopolymer brushes grafted OTCC was obviously less than that of the bare column when the pH value of the mobile phase ranging from 3 to 11. Using the glycopolymer brushes grafted OTCC, the baseline separations of polar molecules and proteins were obtained without peak tailing. The future work will focus on the further development of the glycopolymer brushes for the highly polar compound separation, such as glycans and glycoproteins.
2013, 31(10): 1001-1004
doi: 10.3724/SP.J.1123.2013.03033
Abstract:
The structure of polysaccharide in traditional Chinese medicine (TCM) is complex and characteristic. A method of oligosaccharide electrophoresis analysis assisted with cluster analysis (CA) was established to simultaneously identify TCMs. Six TCMs from three families were selected as experimental subjects and their polysaccharides were extracted. The obtained oligosaccharides via incompletely degraded TCM polysaccharides were derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP). The PMP-oligosaccharide derivatives were separated and analyzed by capillary zone electrophoresis (CZE). The six TCMs were identified by the featured information of oligosaccharide electropherograms with CA. The electrophoresis conditions were as follows: uncoated fused silica capillary column (49 cm/40 cm (total length/effective length)×50 μm); running buffer solution, 50 mmol/L phosphate buffer solution (pH 2.5); detection wavelength, 245 nm; operating voltage, 15 kV; hydrodynamic pressure injection, 10 cm×4 s. The results showed that the six TCMs from three families were effectively identified by the method of TCM oligosaccharide electropherograms combined with CA. This method is a promising tool to identify TCM with good reliability and reproducibility.
The structure of polysaccharide in traditional Chinese medicine (TCM) is complex and characteristic. A method of oligosaccharide electrophoresis analysis assisted with cluster analysis (CA) was established to simultaneously identify TCMs. Six TCMs from three families were selected as experimental subjects and their polysaccharides were extracted. The obtained oligosaccharides via incompletely degraded TCM polysaccharides were derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP). The PMP-oligosaccharide derivatives were separated and analyzed by capillary zone electrophoresis (CZE). The six TCMs were identified by the featured information of oligosaccharide electropherograms with CA. The electrophoresis conditions were as follows: uncoated fused silica capillary column (49 cm/40 cm (total length/effective length)×50 μm); running buffer solution, 50 mmol/L phosphate buffer solution (pH 2.5); detection wavelength, 245 nm; operating voltage, 15 kV; hydrodynamic pressure injection, 10 cm×4 s. The results showed that the six TCMs from three families were effectively identified by the method of TCM oligosaccharide electropherograms combined with CA. This method is a promising tool to identify TCM with good reliability and reproducibility.
2013, 31(10): 1005-1009
doi: 10.3724/SP.J.1123.2013.04045
Abstract:
Gastric cancer is one of the familiar malignant tumors in clinical study. Research on tumor biomarkers has increased noticeably in recent years. An experimental method of protein separation by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was established and improved. A series of effects on capillary electrophoresis (CE) were studied, such as the dynamic coating method of capillary, concentration of polyethylene oxide (PEO) as sieving medium, pH of running buffer, separation voltage, temperature and fluorescent dye. Then the optimized method was established for the determination of gastric cancer tissue and adjacent normal tissue. According to the analysis of the protein fingerprints, the results showed that the similarity of protein was up to 0.8. The molecular masses of differential proteins were almost between 50000 Da and 100000 Da. It indicated that these proteins might be potential biomarkers for cancer diagnosis which may reduce the searching scope. By statistical analysis of histological classification and electrophoresis peak numbers, this method was demonstrated reliably, and had the potential for clinical applications.
Gastric cancer is one of the familiar malignant tumors in clinical study. Research on tumor biomarkers has increased noticeably in recent years. An experimental method of protein separation by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was established and improved. A series of effects on capillary electrophoresis (CE) were studied, such as the dynamic coating method of capillary, concentration of polyethylene oxide (PEO) as sieving medium, pH of running buffer, separation voltage, temperature and fluorescent dye. Then the optimized method was established for the determination of gastric cancer tissue and adjacent normal tissue. According to the analysis of the protein fingerprints, the results showed that the similarity of protein was up to 0.8. The molecular masses of differential proteins were almost between 50000 Da and 100000 Da. It indicated that these proteins might be potential biomarkers for cancer diagnosis which may reduce the searching scope. By statistical analysis of histological classification and electrophoresis peak numbers, this method was demonstrated reliably, and had the potential for clinical applications.
2013, 31(10): 1010-1015
doi: 10.3724/SP.J.1123.2013.03048
Abstract:
A method was developed for the simultaneous determination of tetracyclines (TCs), quinolones (QUs) and sulfadimidine (SM2) in pig manure and chicken dung by a combined high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection coupled with solid phase extraction. The residual antibiotics in manure were extracted with the mixture of methanol, acetic acid and water with the volume ratio of 6:3:1, enriched and purified by a hydrophilic lipophilic balance (HLB) solid-phase extraction cartridge. The C18 chromatographic column was used to complete the separation of the analytes which were detected in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI+). The external standard calibration curves were used for the quantification. All the antibiotics were determined with an excellent linear relationship from 50 to 1000 μg/L for TCs and QUs, and from 5 to 100 μg/L for SM2. The limits of detection for TCs, QUs and SM2 were 0.25-7.18, 0.15-3.16 and 0.04 μg/kg, respectively. The recoveries of TCs, QUs and SM2 in pig manure and chicken dung were 40%-124% at the spiked levels from 0.1 to 10 μg/g (RSDs ranged from 3.0% to 9.5%, n=6). The method was successfully applied to determine the antibiotics in pig manure and chicken dung samples. The method has high accuracy and sensitivity for the determination of TCs, QUs and SM2 in pig manure and chicken dung.
A method was developed for the simultaneous determination of tetracyclines (TCs), quinolones (QUs) and sulfadimidine (SM2) in pig manure and chicken dung by a combined high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection coupled with solid phase extraction. The residual antibiotics in manure were extracted with the mixture of methanol, acetic acid and water with the volume ratio of 6:3:1, enriched and purified by a hydrophilic lipophilic balance (HLB) solid-phase extraction cartridge. The C18 chromatographic column was used to complete the separation of the analytes which were detected in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI+). The external standard calibration curves were used for the quantification. All the antibiotics were determined with an excellent linear relationship from 50 to 1000 μg/L for TCs and QUs, and from 5 to 100 μg/L for SM2. The limits of detection for TCs, QUs and SM2 were 0.25-7.18, 0.15-3.16 and 0.04 μg/kg, respectively. The recoveries of TCs, QUs and SM2 in pig manure and chicken dung were 40%-124% at the spiked levels from 0.1 to 10 μg/g (RSDs ranged from 3.0% to 9.5%, n=6). The method was successfully applied to determine the antibiotics in pig manure and chicken dung samples. The method has high accuracy and sensitivity for the determination of TCs, QUs and SM2 in pig manure and chicken dung.
2013, 31(10): 1016-1020
doi: 10.3724/SP.J.1123.2013.06003
Abstract:
A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of the plant growth regulator (PGR) (paclobutrazol, forchlorfenuron, isopentennyladenine and 6-benzylaminopurine) residues in amphisarcas. The sample was extracted with acetonitrile, then cleaned up by MCX solid phase extraction. The HPLC separation was performed on an Agilent XDB-C18 column with 5 mmol/L ammonium acetate solution and acetonitrile containing 0.1% (volume percentage) formic acid as the mobile phases in a gradient elution mode. The PGRs were determined by MS/MS in positive electrospray ionization mode, and quantified by matrix-matched external standard method. The calibration curves showed good linearity in each range with correlation coefficients greater than 0.999. The limits of quantification (LOQs, S/N>10) were 0.04-1.35 μg/kg and the limits of detection (LODs, S/N>3) were 0.01-0.41 μg/kg for the four PGRs spiked in cucumber and apple. The recoveries of the four PGRs spiked at three levels ranged from 81.0% to 93.3%, with the relative standard deviations (RSDs) of 3.5%-9.5%. The sensitivity, accuracy and precision of the method meet the technical standards of the pesticide determination. Therefore the method can be applied to the determination of the four PGRs in amphisarcas.
A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of the plant growth regulator (PGR) (paclobutrazol, forchlorfenuron, isopentennyladenine and 6-benzylaminopurine) residues in amphisarcas. The sample was extracted with acetonitrile, then cleaned up by MCX solid phase extraction. The HPLC separation was performed on an Agilent XDB-C18 column with 5 mmol/L ammonium acetate solution and acetonitrile containing 0.1% (volume percentage) formic acid as the mobile phases in a gradient elution mode. The PGRs were determined by MS/MS in positive electrospray ionization mode, and quantified by matrix-matched external standard method. The calibration curves showed good linearity in each range with correlation coefficients greater than 0.999. The limits of quantification (LOQs, S/N>10) were 0.04-1.35 μg/kg and the limits of detection (LODs, S/N>3) were 0.01-0.41 μg/kg for the four PGRs spiked in cucumber and apple. The recoveries of the four PGRs spiked at three levels ranged from 81.0% to 93.3%, with the relative standard deviations (RSDs) of 3.5%-9.5%. The sensitivity, accuracy and precision of the method meet the technical standards of the pesticide determination. Therefore the method can be applied to the determination of the four PGRs in amphisarcas.
2013, 31(10): 1021-1027
doi: 10.3724/SP.J.1123.2013.03019
Abstract:
An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analytical method was established for the simultaneous determination of 24 forbidden industrial dyes in grain and meat products. The sample was extracted with methanol and acetonitrile, and cleaned-up by a WAX solid phase extraction column. The solution was separated on an ACQUITY UPLC BEH C18 column eluted with a mixture of 10 mmol/L ammonium acetate-0.2%formic acid aqueous solution and methanol-acetonitrile (7:3, v/v) as the mobile phases, and then analyzed in multiple reaction monitoring (MRM) mode. The correlation coefficients were above 0.99, the average recoveries were 61%-116%, and the relative standard deviations (RSD, n=6) were lower than 13%. The quantification limits were 0.1-4.0 μg/kg. This method is simple, effective, sensitive, and suitable for the determination and confirmation of the 24 forbidden industrial dyes in grain and meat products.
An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analytical method was established for the simultaneous determination of 24 forbidden industrial dyes in grain and meat products. The sample was extracted with methanol and acetonitrile, and cleaned-up by a WAX solid phase extraction column. The solution was separated on an ACQUITY UPLC BEH C18 column eluted with a mixture of 10 mmol/L ammonium acetate-0.2%formic acid aqueous solution and methanol-acetonitrile (7:3, v/v) as the mobile phases, and then analyzed in multiple reaction monitoring (MRM) mode. The correlation coefficients were above 0.99, the average recoveries were 61%-116%, and the relative standard deviations (RSD, n=6) were lower than 13%. The quantification limits were 0.1-4.0 μg/kg. This method is simple, effective, sensitive, and suitable for the determination and confirmation of the 24 forbidden industrial dyes in grain and meat products.
2013, 31(10): 1028-1032
doi: 10.3724/SP.J.1123.2013.03006
Abstract:
An analytical method for the determination of nitroxynil, oxyclozanide, closantel and rafoxanide in liquid milk by high performance liquid chromatography (HPLC) has been established. The milk sample was extracted with acetonitrile containing 1% (v/v) triethylamine. The supernatant was purified by an anion exchange solid phase extraction column. The analyte was detected by ultraviolet detector after the HPLC separation on a C18 RP column. The mobile phase was composed of acetonitrile-0.02 mol/L ammonium acetate solution with pH 4.0. The linear ranges of the four drugs in the spiked blank milk samples were 5-500 μg/kg, and the correlation coefficients were higher than 0.99. The limits of detection (LOD) were 3 μg/kg, and the limits of quantification (LOQ) were 5 μg/kg. The average recoveries and relative standard deviations (RSDs) of nitroxynil, oxyclozanide, closantel and rafoxanide at the spiked levels of 1/2MRL (maximum residue limit), MRL and 2MRL ranged from 92.20% to 96.13%, 5.55% to 16.30%; 87.40% to 94.74%, 5.40% to 12.21%; 86.97% to 91.09%, 2.67% to 8.17%; and 77.86% to 95.36%, 5.02% to 13.15% respectively. The method is simple and sensitive for the quantification of phenolic and salicylanilide anthelmintics in liquid milk.
An analytical method for the determination of nitroxynil, oxyclozanide, closantel and rafoxanide in liquid milk by high performance liquid chromatography (HPLC) has been established. The milk sample was extracted with acetonitrile containing 1% (v/v) triethylamine. The supernatant was purified by an anion exchange solid phase extraction column. The analyte was detected by ultraviolet detector after the HPLC separation on a C18 RP column. The mobile phase was composed of acetonitrile-0.02 mol/L ammonium acetate solution with pH 4.0. The linear ranges of the four drugs in the spiked blank milk samples were 5-500 μg/kg, and the correlation coefficients were higher than 0.99. The limits of detection (LOD) were 3 μg/kg, and the limits of quantification (LOQ) were 5 μg/kg. The average recoveries and relative standard deviations (RSDs) of nitroxynil, oxyclozanide, closantel and rafoxanide at the spiked levels of 1/2MRL (maximum residue limit), MRL and 2MRL ranged from 92.20% to 96.13%, 5.55% to 16.30%; 87.40% to 94.74%, 5.40% to 12.21%; 86.97% to 91.09%, 2.67% to 8.17%; and 77.86% to 95.36%, 5.02% to 13.15% respectively. The method is simple and sensitive for the quantification of phenolic and salicylanilide anthelmintics in liquid milk.
