Capillary gas-chromatographic determination of spermidine in hair lotion

Biogenic polyamines, such as spermidine (SPD, NH2-(CH2)4-NH-(CH2)3-NH2), are ubiquitous polycationic molecules which play a definitive role in many biological processes such as nucleic acid metabolism, protein synthesis, and cell growth. SPD is commonly used as an ingredient in hair lotions, because it seems to promote hair growth. This work describes a capillary GC method for quantitative determination of SPD in hair lotions using 1,6-diaminohexane as internal standard, a methyl silicone capillary column, and a flame ionisation detector. Aliquots of hair lotion were treated with an alkaline aqueous solution and internal standard was added. The emulsion was extracted with diethyl ether containing ethyl chloroformate. Ether extracts, evaporated to dryness and reconstituted in ethyl acetate, were analysed by capillary GC with flame ionisation detection. Validation took into account the specificity, linearity, precision, and accuracy of the analytical method: these parameters were valid for the quantitative determination of SPD in hair lotion.     

Rapid,sensitive and simultaneous determination of fluorescence-labeled designated substances controlled by the Pharmaceutical Affairs Law in Japan by ultra-performance liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 “designated substances” (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC™ BEH C₁₈ column (1.7 μm, 100 mm × 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile–methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H]⁺ obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection ( \textS/N = 3 ) \left( {{\text{S/N}} = 3} \right) and the limits of quantification ( \textS/N = 10 ) \left( {{\text{S/N}} = 10} \right) in 0.1 mL of human plasma and urine for the present method were 0.30–150 pmol and 1.0–500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products.      

Sensitive Laser induced fluorescence detection of Polyamine-Fluoresceinisothiocyanate-derivatives after capillary zone electrophoretic separation

Capillary zone electrophoresis (CZE) has been applied for the separation and quantification of the polyamines putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), after their derivatization with the fluoresceinisothiocyanate isomer I (FITC).The derivatization conditions (character of the derivatization medium and concentration, organic modifier, pH and temperature) and CZE operation conditions have been optimized with respect to a sensitive Laser induced fluorescence detection (_excitation=488 nm/_detection=520 nm). A complete separation of CAD from PUT was impossible under the conditions chosen. The detection limits and relative standard deviations for SPM, SPD, CAD, PUT were determined to be 2.9 (12%), 3.2 (7.6%), 0.7 (4.6%), 1.2 (7.6%) nmol/L, respectively. The method has been applied to a pine needle extract.List of abbreviations a intercept - b slope - b_norm normalized slope of the calibration plot - CAD cadaverine - CZE capillary zone electrophoresis - DL detection limit - EOF electroosmotic flow - FITC fluoresceinisothiocyanate - FMOC 9-fluorenylmethyl chloroformate - HEC hydroxyethyl-cellulose - LIF Laser induced fluorescence - MW molecular weight - OPA o-phthalaldehyde - P/ACE programmable/automatical capillary electrophoresis - PUT putrescine - q charge - RSD relative standard deviation - SDS sodium dodecylsulfate - SPD spermidine - SPM spermine - t_migr migration time - w basic peak width - _eff effective mobility     

A quantitative GC‐MS method for three major polyamines in postmortem brain cortex

A quantitative method for putrescine (PUT), spermidine (SPD) and spermine (SPM) in homogenized postmortem human brain tissue is described that employs a novel, simple and rapid extractive derivatization with ethylchloroformate and trifluoroacetylation. These amines are metabolites of ornithine and are metabolically interconvertible in mammals. The method was developed to support an ongoing epidemiological study correlating these amines with the frequency of suicide. The isolation methodology is robust and requires less work and time than many previous methods. Analysis is by conventional electron ionization GC‐MS with selected ion monitoring using a stable isotope‐labeled analog for PUT and a chemical analog for SPD and SPM as internal standards. The time required for chromatographic analysis, about 20 min, is determined by the wide range of the relative volatilities of the derivatized polyamines. The method allows the quantitation of PUT down to 10 ng/g and SPD and SPM down to 100 and 1000 ng/g, respectively of wet tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection

There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE‐LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4‐chloro‐7‐nitro‐2,1,3‐benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70°C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive‐MS with an ESI source was used for identification of the bis‐derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra‐ and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples.     

A 4-hydroxy-N′-[(E)-(2-hydroxyphenyl)methylidene]benzohydrazide-based sorbent material for the extraction-HPLC determination of biogenic amines in food samples

A sorbent material based on a newly synthesized hydrazone ligand, 4-hydroxy-N′-[(E)-(2-hydroxyphenyl)methylidene]benzohydrazide was prepared by immobilizing the ligand into a silica sol-gel matrix. The capability of the sorbent material for the extraction of seven biogenic amines (BAs), i.e., tryptamine (TRY), β-phenylethylamine (PEA), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), and spermidine (SPD) was studied. Under the adopted conditions, the sorbent showed good selectivity towards PUT, CAD, HIS and SPD (% extraction (%E) > 96) while %E for TYR, TRY and PEA were 82.0, 78.9 and 46.4%, respectively. The sorbent could be used up to six extraction cycles for SPD, CAD and PUT and was applied to the determination of food samples (“budu”, ketchup, orange juice, soy sauce) that were spiked with 20 mg L⁻¹ of the BAs. The extracted analytes were derivatized with dansyl chloride before the HPLC determination. With the exception of HIS and TYR in “budu” sample, reasonable recoveries were found for the other analytes in all the tested food samples.     

Automated determination of polyamines by high-performance liquid chromatography with simple sample preparation

Recently, a new fully endcapped reversed-phase packing material, Inertsil, was introduced, especially suitable for the determination of basic compounds. We used this packing material to separate o-phthaldialdehyde (OPA) derivatives of amino acid derivatives completely from the OPA derivatives of spermine (SPM), spermidine (SPD), putrescine (PUT) and cadaverine (CAD). The obtained separation made the commonly used off-line extraction procedure redundant and thus an on-line sample clean-up was introduced. This enabled automation of the procedure resulting in a better reproducibility and a more efficient use of equipment. Furthermore, no studies are required to determine the extraction recovery.

The present method has a cycle time of 30 min. A linear response for each polyamine was found up to 250 pmol, with an R² ranging from 0.9981 (SPM) to 0.9998 (CAD). The limit of detection, calculated at a signal-to-noise ratio of 3, was 0.1 pmol, corresponding to a plasma concentration of 0.1 μmol/l. The coefficient of variation (CV) for the peak area was below 3% and for retention times below 0.5% (n=15).

In order to evaluate the applicability of the method, three different types of sample were chromatographed, e.g. urine (obtained from healthy human volunteers), pig plasma and sulfosalicylic acid homogenates of pig intestine biopsies. Tissue homogenates and urine-specimen could easily be quantitated, while plasma concentrations were just above the limit of detection, resulting in a plasma CV ranging from 4.8% (SPM) to 13.6% (SPD) and a tissue CV ranging from 2.1% (SPM) to 8.5% (CAD), The urinary CVs were not determined.

In conclusion, the present method provides an easy way to measure polyamine concentrations for most applications.     

Rapid determination and confirmation of biogenic amines in tuna loin by gas chromatography/mass spectrometry using ethylchloroformate derivative

A gas chromatography-mass spectrometry (GC/MS) method is described for the easy rapid determination and simultaneous confirmation of the biogenic amines putrescine (PUT), cadaverine (CAD), histamine (HTA), and spermidine (SPD) in fresh frozen tuna loin. The method can also be used to monitor tyramine (TYR). The method involves homogenization of fish tissue, extraction of biogenic amines into trichloroacetic acid solution, centrifugation, alkalization, and derivatization of supernatant with ethylchloroformate. All seafood species were fortified to contain 2.5, 5.0, 10.0, 12.5, and 25.0 microg/g (ppm) PUT, CAD, and SPD; and 10.0, 20.0, 40.0, 50.0, and 100.0 microg/g (ppm) HTA. Determination was based on standard curves for each analyte using peak areas with matrix standards equivalent to a concentration range bracketing the spike level. A set of 5 matrix controls (unfortified tuna tissue) was also analyzed; only endogenous SPD was found in all samples. The interassay average recoveries ranged from 57 to 79% across analytes and spike levels.     

超高效液相色谱-串联质谱法测定草鱼肉中阿苯达唑及其代谢物残留

建立了同时测定草鱼肉中阿苯达唑及其3种代谢物阿苯达唑亚砜、阿苯达唑砜、阿苯达唑-2-氨基砜的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。草鱼肉样品通过碱性乙酸乙酯提取,正己烷净化,超高效液相色谱分离,串联质谱检测,氘代同位素内标定量。本方法分析时间短,在4 min内即可完成4种被分析物的液质联用分析。本方法在0.1～10μg/kg范围内各物质线性良好,线性系数在0.9991～0.9996之间。阿苯达唑、阿苯达唑亚砜、阿苯达唑砜、阿苯达唑-2-氨基砜在0.5～5.0μg/kg添加水平下的平均回收率分别为98.4%～102.6%,97.1%～103.4%,98.8%～103.7%和101.1%～104.2%检出限分别为0.2,0.1,0.2和0.1μg/kg;日内精密度小于4.77%,日间精密度小于3.03%。本方法为阿苯达唑及其代谢物的检测及监控提供了一种快速、灵敏度高、重现性好的定量分析方法。     

Simultaneous determination of L-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method: application to human urine samples

We have developed and described a highly sensitive, accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle in human urine samples. After pre-column derivatization with ortho-phthaldialdehyde (OPA) reagent containing 3-mercaptopropionic acid (3MPA), the fluorescent derivatives were separated by a gradient elution and detected by fluorescence measurement at 338 nm (excitation) and 455 nm (emission). l-Arginine (ARG) and its metabolites: l-glutamine (GLN), N^G-hydroxy-l-arginine (NOHA), l-citrulline (CIT), N^G-monomethyl-l-arginine (NMMA), l-homoarginine (HARG), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), symmetric N^G,N^G′-dimethyl-l-arginine (SDMA), l-ornithine (ORN), putrescine (PUT), agmatine (AGM), spermidine (SPERMD) and spermine (SPERM) were extracted in a cation-exchange solid-phase extraction (SPE) column and after derivatization separated in a Purospher^® STAR RP-18e analytical column. The calibration curves of analysed compounds are linear within the range of concentration: 45-825, 0.2-15, 16-225, 12-285, 0.1-32, 15-235, 0.1-12, 0.1-12, 10-205, 0.02-12, 0.1-24, 0.01-10 and 0.01-8 nmol mL⁻¹ for GLN, NOHA, CIT, ARG, NMMA, HARG, ADMA, SDMA, ORN, PUT, AGM, SPERMD and SPERM, respectively. The correlation coefficients are greater than 0.9980. Coefficients of variation are not higher than 6.0% for inter-day precision. The method has been determined or tested for limits of detection and quantification, linearity, precision, accuracy and recovery. All detection parameters of the method demonstrate that it is a reliable and efficient means of the comprehensive determination of ARG and its 12 main metabolites, making this approach suitable for routine clinical applications. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.     

Reversed-phase liquid chromatographic separation and simultaneous fluorimetric detection of polyamines and their monoacetyl derivatives in human and animal urine, serum and tissue samples: an improved, rapid and sensitive method for routine application

A highly sensitive and precise method for the determination of the polyamines putrescine, cadaverine, spermidine and spermine and all their monoacetyl derivatives in a single analysis in human and animal urine, serum and tissue samples is described. For polyamine separation, an ion-pairing reversed-phase high-performance liquid chromatographic (HPLC) method is used, followed by post-column derivatization with o-phthalaldehyde and consecutive fluorescence detection. Urine and serum samples are purified with a Bond Elut silica cartridge. The detection limit for polyamines is 0.5-1.0 pmol and excellent linearity is achieved in the range from 3 pmol up to more than 10 nmol. The influence of some modifications of different analytical steps such as the temperature of the HPLC column and the derivatization reaction coil and the o-phthalaldehyde flow-rate is described. Quality control data and measurements of the reproducibility of the method are presented. In order to establish a rapid analytical method for easy routine use, all steps for preparation and quantitative analysis are minimized. This method was applied to the determination of total polyamines in human urine and serum hydrolysate and of free and acetylated polyamines in human urine and pancreatic tissue of the rat. Values for normal polyamine concentrations in the urine and serum of fifteen male and fifteen female healthy volunteers and in the pancreas of ten normal rats are presented.     

Assessment of the capillary zone electrophoretic behavior of proteins in the presence of electroosmotic modifiers: protein-polyamine interaction studied using a polyacrylamide-coated capillary

The use of polyamines as electroosmotic modifiers has been shown to be effective in enhancing resolution of protein glycoforms in capillary zone electrophoresis (CZE) using a bare capillary tube. In this study, effectiveness was evaluated by using a polyacrylamide-coated capillary tube instead of a bare capillary tube. Electropherograms obtained in the presence of polyamines were inferior to those obtained in their absence with respect to resolution. Electrophoretic mobility of the proteins decreased and their peaks were broadened by polyamines bound to them. This unfavorable effect was dependent on both the species of polyamines and the pH values of the electrolyte buffer. The reduction of resolution caused by polyamines was in the following order: spermidine (SPD) approximately spermidine-tri-hydrochloride (SPD-HCI) > putrescine (PUT) > hexamethonium chloride (HMC). The observed effect can be ascribed to the formation of complexes between the proteins and the polyamines. In addition, for the bare capillary tube the complexes showed interaction with the inner surface, resulting in local suppression of electroosmosis and poor resolution. The high resolution obtained in the coated capillary tube was reduced in the presence of the polyamines. Thus, the use of the polyamines has a negative effect on the analysis of protein microheterogeneity as a result of protein-polyamine interaction.     

Mass fragmentographic identification of polyamine metabolites in the urine of normal persons and cancer patients, and its relevance to the use of polyamines as tumour markers

The mass fragmentographic identification of N-(2-carboxyethyl)-4-amino-n-butyric acid, N-(3-aminopropyl)-N1-(2-carboxyethyl)-1,4-diaminobutane, N,N1-bis(2-carboxyethyl)-1,4-diaminobutane, and delta-aminovaleric acid in acid-hydrolysed urines of a normal person and two cancer patients is described. A previous study, in which the metabolic fate of intraperitoneally injected polyamines in rats was investigated, revealed that these compounds should be considered as non-alpha-amino acid metabolites of the naturally occurring polyamines. Quantification of polyamines and their non-alpha-amino acid metabolites by gas chromatography with nitrogen--phosphorus detection showed that, relative to the parent polyamines, humans normally excrete higher quantities of polyamine catabolites in urine than rats, suggesting that humans catabolize polyamines more efficiently. As illustrated by the follow-up of the concentrations of polyamines and their catabolites in the urine of a patient with high-grade non-Hodgkin lymphoma during chemotherapy, the catabolic pressure on polyamines may be considerably increased during neoplastic diseases, since an even higher proportion of oxidized polyamine metabolites was observed. It is therefore suggested that the additional measurement of the circulating concentrations of polyamine-degrading enzymes is of importance for the correct interpretation of polyamine (metabolite) determinations for oncological purposes.     

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC‐ESI‐MS/MS

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole derivatives and liquid chromatography–electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N¹‐acetylputrescine (N¹‐actPUT), N⁸‐acetylspermidine, N¹‐acetylspermine, 1,3‐diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal‐to‐noise ratio 5) were 5–100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6‐diaminohexane, vs the injected amounts of polyamines (r² > 0.996), and the intra‐day and inter‐day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14–110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N¹‐actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid analysis of N‐linked oligosaccharides in glycoproteins (ovalbumin,ribonuclease B and fetuin) by reversed‐phase ultra‐performance liquid chromatography with fluorescence detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid, selective and sensitive determination of N‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra‐performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). The asparaginyl‐oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel‐permeation chromatography were labeled with 1‐pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF‐MS. The PSC‐labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI‐TOF‐MS. As the results, 15, eight and four kinds of N‐linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N‐linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd.     

Rapid separation and identification of phenolic and diterpenoid constituents from Radix Salvia miltiorrhizae by high-performance liquid chromatography diode-array detection, electrospray ionization time-of-flight mass spectrometry and electrospray ionization quadrupole ion trap mass spectrometry

High-performance liquid chromatography-diode array detection (HPLC-DAD), electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and electrospray ionization quadrupole ion trap mass spectrometry (HPLC-ESI-MSn) were used for the isolation, identification and structural analysis of water-soluble phenolic and nonpolar diterpenoid constituents in Dan-shen (Radix Salvia miltiorrhizae) which was prepared by sonication in 70% methanol. Mass spectra were obtained by ESI-TOF-MS and electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). A formula database of known constituents in Dan-shen was established and most constituents were rapidly identified by HPLC-DAD/ESI-TOF-MS by matching their accurate molecular masses with the formulae of the compounds in the database. Compounds with the same molecular formula could not be differentiated by TOF-MS; however, QIT-MS could differentiate those compounds and elucidate their structures based on their characteristic fragmentation. HPLC-DAD, HPLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary information for the identification of the constituents in Dan-shen. Forty constituents were identified in 30 min based on their positive and negative ion ESI mass spectra and liquid chromatographic information. Thus the method described is useful for the rapid analysis of multiple constituents in Dan-shen.     

Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species

Steroidal alkaloids are naturally occurring nitrogen-containing compounds in many edible or medicinal plants, such as potato, tomato, Fritillaria and American hellebore, which possess a variety of toxicological and pharmacological effects on humans. The aim of this study is to explore the potential of liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) method in the determination of these important alkaloids in plant matrices. The application of this method has been proven through 26 naturally occurring steroidal alkaloids in Fritillaria species. Accurate mass measurements within 4 ppm error were obtained for all the alkaloids detected out of various plant matrices, which allowed an unequivocal identification of the target steroidal alkaloids. The bunching factor for mass spectrometer, an important parameter significantly affecting the precision and accuracy of quantitative method, was firstly optimized in this work and satisfactory precision and linearity were achieved by the optimization of that parameter. The ranges of RSD values of intra-day and inter-day variability for all alkaloids were decreased remarkably from 41.8-159% and 13.2-140% to 0.32-7.98% and 2.37-16.1%, respectively, when the value of bunching factor was optimized from 1 to 3. Linearity of response more than two orders of magnitude was also demonstrated (regression coefficient >0.99). The LC/TOF-MS detection method offered improvements to the sensitivity, compared with previously applied LC (or GC) methods, with limits of detection down to 0.0014-0.0335 microg/ml. The results in this paper illustrate the robustness and applicability of LC/TOF-MS for steroidal alkaloids analysis in plant samples. In addition, relative quantitative determination of steroidal alkaloid with one popular analyte verticinone which is commercially available was also investigated in order to break through the choke point of lack of standards in phytochemical analysis. The accuracies of relative quantitative method for steroidal alkaloids determinations with verticinone were 90.6-110.0% (average 98.5%) suggesting that it is feasible to quantify steroidal alkaloids by the proposed relative quantitative determination method within acceptable errors.     

SiW11掺杂二氧化硅纳米纤维(SiW11/SiO2)的制备及对拟南芥叶中多胺的萃取分析

采用静电纺丝技术制备了SiW₁₁掺杂的二氧化硅纳米纤维(SiW₁₁/SiO₂)材料, 其中掺杂的SiW₁₁质量占纤维质量的7.83%, 制备的SiW₁₁/SiO₂材料尺寸均一, 在考察的pH范围内带负电, 与未掺杂SiW₁₁的二氧化硅纳米纤维相比, SiW₁₁/SiO₂材料表现出强阳离子交换作用, 能有效萃取尸胺和腐胺. 在最优条件下, 建立了注射器分散固相萃取(In syringe dSPE)-超高效液相色谱-串联质谱联用(UHPLC-MS/MS)检测拟南芥样品中多胺的方法. 结果表明, 尸胺和腐胺分别在10~1000和20~1000 ng/mL浓度范围内具有良好的线性关系(R²≥0.9950), 检出限和定量限分别为0.5~0.9和1.6~3.0 ng/mL. 将该方法应用于拟南芥样品中尸胺和腐胺的检测, 加标回收率在87.5%~111.3%之间, 相对标准偏差RSD<5.0%. 建立的SiW₁₁/SiO₂制备方法解决了多金属氧酸盐(POM)在修饰改性过程中存在的制备繁琐、 固载量少的问题, 拓宽了POM在分离领域中的应用.     

Sensitive fluorimetric method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles. The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 +/- 3.1, and spermine 8.4 +/- 2.8 nmoles/ml packed erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml. The polyamine content of the erythrocytes from patients with malignant neoplasms was significantly elevated.