高效液相色谱荧光测定及质谱鉴定分析血清中胆汁酸

利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基苯磺酸酯(BDEBS)作柱前衍生化试剂,在EclipseXDB-C8色谱柱上,采用梯度洗脱对10种胆汁酸(BAs)荧光衍生物进行了优化分离。95℃下在二甲基亚砜(DMSO)溶剂中以柠檬酸钾作催化剂,衍生反应35min后获得稳定的荧光产物,衍生反应完全。荧光激发和发射波长分别为λex=333nm,λem=390nm。采用大气压化学电离源(APCISource)正离子模式,实现了血清中胆汁酸的定性测定。分析物的定量测定采用荧光法进行。线性回归系数均在0.9996以上,检出限为22.36~44.57×10-15mol。     

Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent

A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid-liquid extraction using a mixture of ethyl acetate-diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min⁻¹. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05-16.00 μg mL⁻¹ for nateglinide with a limit of quantitation of 0.05 μg mL⁻¹. Quality control samples (0.05, 4.50 and 16.00 μg mL⁻¹) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Pre-column fluorescence derivatization of peptides containing arginine aldehyde moiety in high-performance liquid chromatography

Summary A sensitive reversed-phase high-performance liquid chromatographic method with pre-column fluorescence derivatization has been developed for the determination of DLL-MePhe-Pro-Arg-H (LY-DLL, LY-294468). In the search for a derivatization method for the determination of the tripeptide aldehyde, which is present in equilibrium structures in aqueous solution, it was found that the guanidino group of the argininal residue was not converted into a fluorescent derivative by reaction with benzoin. However, if the LY-DLL was first converted into a LY-DLL-TRIS adduct, a fluorescent product could be obtained by the reaction of LY-DLL with TRIS in TRIS(HCl) buffer (pH 8.5) followed by benzoin in the presence of beta-mercaptoethanol and sodium sulphite in an alkaline medium.     

Residue determination of glyphosate in environmental water samples with high-performance liquid chromatography and UV detection after derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

A pre-column derivatization high-performance liquid chromatographic method for glyphosate analysis has been developed. Derivatization of glyphosate was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). In pH 9.5 H₃BO₃-Na₂B₄O₇ media, the reaction of glyphosate with CNBF completed at 60 °C for 30 min. The labeled glyphosate was separated on a Kromasil C18 column (250 mm × 4.6 mm, 5 μm) at room temperature and UV detection was applied at 360 nm. The separation of labeled glyphosate was achieved within 15 min by gradient elution mode. Compared to other pre-column derivatization, this derivatization was performed more mildly, the derivative was more stable, and the detection limits of a few reagents were higher than CNBF, except 9-fluorenylmethyl chloroformate (FMOC-Cl) using fluorescence and mass spectrometry, however, this reagent avoid to be removed after derivatization like FMOC-Cl. The detection limit of glyphosate was 0.009 mg L⁻¹ (S/N = 3) without preconcentration and reach MRL, which is set at the level of 0.1 mg L⁻¹ in China. The method linearity correlation coefficient was 0.9999, in concentrations ranging from 0.3 to 48.5 mg L⁻¹. The proposed method has been applied to the quantitative determination of glyphosate in environmental water with recoveries of 91.80-100.20% and R.S.D. of 2.27-6.80, depending on the sample investigated.     

Determination of glutathione in pharmaceuticals and cosmetics by HPLC with UV and fluorescence detection

Summary A selective HPLC determination of reduced glutathione (GSH) in pharmaceutical and cosmetic formulations is described based on prechromatographic derivatization with 4-(6-methylnaphthalen-2-yl)-4-oxo-2-buteneoic acid. The derivatization reaction is rapid under mild reaction conditions (10 min at ambient temperature and pH 7.5) and the excess reagent can be removed by liquid-liquid extraction. The thiol adducts were chromatographed on a C-8 column using 0.05 M triethylammonium phosphate (pH 4.0) — acetonitrile 68∶32 (v/v), as the mobile phase; UV and fluorescence detection (l_em 450 nm, l_exc 300 nm) were both used. The structure of the thiol adducts was confirmed by¹H and¹³C NMR spectra using sodium methanethiolate as thiol nucleophile. This work constitutes part of the thesis for the “Dottorato di ricerche” of Dr. Roberto Gotti.     

Determination of sugars by liquid chromatography with post-column catalytic derivatization and fluorescence detection

Summary A method for the determination of reducing sugars such as fructose and glucose and nonreducing sugar such as sucrose by high performance liquid chromatography followed by an acidic hydrolysis and a derivatization with benzamidine has been developed. After separation of sugars on a gel column packed with a polymer-based cation exchange material (Sugar-Pak I, Waters-Millipore), the sucrose is first hydrolysed in a solid phase reactor to convert it into reducing subunits. A post-column fluorigenic reaction with benzamidine under alkaline condition allows the selective determination of both natural and converted reducing carbohydrates.This procedure has proven to be selective (fluorigenic detection) and highly sensitive (allowing detection as little as picomoles amounts), reproducible and linear over a broad range of concentrations: 5×10^–4 to 1.0×10^–2 M.The applicability of this method to natural matrices such as plant extracts and beverages is also described. The sugar content of a barley extract has been determined and compared with a specific enzymatic test. The determined sugar content of natural and commercial lemon juices as well as of Cola beverages has been compared with those found by the conventional LC refractive index analytical procedure. In all cases, the results were comparable and were within the experimental errors of the methods.     

Simultaneous quantification of five anthraquinones in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive high-performance liquid chromatographic method with fluorescence detection (excitation 435 and emission 515 nm) was established and validated for quantification of five anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) in rat plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (1,8-dihydroxyanthraquinone) were separated on a reversed-phase C(18) column with water-phosphoric acid-methanol as mobile phase at a flow rate of 1 mL/min. The linear ranges of the calibration curves were 6.5-1300 ng/mL for aloe-emodin, 20-4000 ng/mL for rhein, 40-8000 ng/mL for emodin, 15-3000 ng/mL for chrysophanol and 13-2600 ng/mL for physcion. The lower limit of quantification was 6.5 ng/mL for aloe-emodin, 20 ng/mL for rhein, 40 ng/mL for emodin, 15 ng/mL for chrysophanol and 13 ng/mL for physcion. The mean accuracy was 94.3-105.1% for aloe-emodin, 90.3-108.8% for rhein, 92.6-106.7% for emodin, 95.8-103.8% for chrysophanol and 98.7-101.2% for physcion. The within-batch and between-batch precisions were < or = 5.5% and < or = 13.4%, respectively. This method is suitable for determining the five anthraquinones in plasma simultaneously and thus investigating the pharmacokinetics of anthraquinones from Xiexin decoction in rats.     

Sub-nanogram detection of dihydroartemisinin after chemical derivatization with diacetyldihydrofluorescein followed by high-performance liquid chromatography and UV absorption

Summary The use of diacetyldihydrofluorescein (DADF) for derivatization of dihydroartemisinin (dihydroqinghaosu, DHQHS) is proposed. The reaction between DHQHS and this reagent in the presence of 4-dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) was complete in 8 hours at room temperature giving about 80 per cent theoretical yield. The derivative showed intense UV absorption, thus providing a sensitivity of 0.1 nanogram by UV detection after column separation. The influences of the ratio of the reagents, reaction temperature, chromatographic conditions and the extent of detection linearity were investigated. The reaction gave consistent results and chromatographic separation was not affected by an excess of the reagent or side products.     

液相微萃取-高效液相色谱法分析葡萄汁中多酚类化合物

建立了一种基于液相微萃取与高效液相色谱联用技术测定葡萄汁中鞣花酸、白藜芦醇和槲皮素的分析方法. 比较了单液滴液相微萃取和中空纤维液相微萃取两种萃取模式, 选择了单液滴液相微萃取作为3种多酚类化合物的液相微萃取模式. 考察了搅拌速度、萃取时间、料液相pH和料液相离子强度的影响. 鞣花酸、白藜芦醇和槲皮素的富集倍数分别为48.4、 79.4和155.8, 方法的线性范围为0.0050～5.0 μg/mL, 鞣花酸、白藜芦醇和槲皮素的检出限分别为0.015, 0.0020, 0.0080 μg/mL, 相对标准偏差分别为2.0%, 1.8%和1.7%. 用于实际样品葡萄汁的分析, 加标回收率在81.9%～102.3%之间.     

高效液相色谱荧光检测法测定药物中的氯乙酰氯

建立测定药物中氯乙酰氯含量的高效液相色谱–荧光检测方法。以吖啶酮乙酰肼为荧光标记试剂,对氯乙酰氯进行柱前衍生。在室温下反应15 min,衍生产率达到最大。衍生溶液在XDB–C18柱上,以水和乙腈为流动相进行分析,激发波长和发射波长分别为255 nm和429 nm。氯乙酰氯浓度在1~1 000 nmol/L范围内与色谱峰面积具有良好的线性关系,线性相关系数r=0.999 9。方法的检出限为0.35 nmol/L,仪器精密度和方法精密度分别为0.52%和0.67%(n=6)。样品加标回收率为92.5%~95.6%。该方法简单、准确,精密度良好,可用于测定药物中氯乙酰氯的残留量。     

Determination of carbendazim and thiabendazole in apple juice by hollow fibre-based liquid phase microextraction-high performance liquid chromatography with fluorescence detection

A hollow fibre-based liquid phase microextraction (HF-LPME) coupled with high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed for the determination of carbendazim (MBC) and thiabendazole (TBZ) in apple juice. Some important extraction parameters, such as the pH of the sample solution, extraction time, stirring rate and salt concentration, were optimised. As a result, the optimal HF-LPME conditions were selected as follows: A 4?mL of sample solution (donor phase) at pH of 7.5 was used for the extraction. The pores of the hollow fibre were impregnated by 1-octanol, and 5?mM HCl (pH?=?2.5) was used as the extraction solvent. The extractions were conducted at a stirring rate of 800?rpm for 40?min. After extraction, 10?µL of the extraction solvent was injected into the HPLC system for analysis. The average enrichment factors were 106 and 114 for MBC and TBZ, respectively. A good linear relationship existed in the range of 2.5?～?500?µg?L^?1 and 5?～?500?µg?L^?1for MBC and TBZ in apple juice with the correlation coefficients (r) of 0.9995 and 0.9991, respectively. The limits of detection were 0.8?µg?L^?1 for MBC and 1.5?µg?L^?1 for TBZ (S/N?=?3?:?1). The recoveries of the method were between 86.3% and 106.0% with the relative standard deviations (RSDs) ranging from 3.3% to 8.5%. The HF-LPME-HPLC method has been successfully applied to the analysis of MBC and TBZ in apple juice, indicating that LPME-HPLC may be a promising combination for the analysis of pesticide residues for some food samples.     

Determination of trans-fatty acids in food samples based on the precolumn fluorescence derivatization by high performance liquid chromatography

Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this precolumn fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography with fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25°C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C₁₈ column with isocratic elution in 14 min. With fluorescence detection at λ_ex/λ_em = 490 /510 nm, the linear range of the TFAs was 1.0-200 nM with low detection limits in the range of 0.1–0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02 to 109.22% with low relative standard deviations of 1.2–4.3% (n = 6).     

柱前衍生高效液相色谱-荧光检测法测定血浆中同型半胱氨酸

建立了一种柱前衍生高效液相色谱-荧光检测法用于测定血浆中同型半胱氨酸(Hcy)。使用三(2-羧乙基)膦盐酸盐(TCEP)为还原剂,N-(1-芘)马来酰亚胺(NPM)为衍生剂进行样品预处理,Agilent Hypersil C-18柱(250 mm×4.0 mm, 5 μm)进行分离,流动相为15 mmol/L醋酸钠-乙腈-混合酸(300 mL水中含1 mL醋酸和1 mL磷酸)混合溶液,采用梯度洗脱,荧光检测激发波长为330 nm,发射波长为380 nm。Hcy的回收率为(102.08±4.94)%。线性范围为0.500～100 μmol/L,检出限（以信噪比为3计）为0.016 μmol/L。日内与日间相对标准偏差均小于5%。利用该方法对7例高血压患者和7例健康志愿者的血浆进行了测定,结果表明两组间的Hcy含量存在显著的差异(p＜0.05)。本方法简单、快速、灵敏、特异,适用于血浆Hcy的临床定量测定。     

Selective and sensitive determination of lipoyllysine (protein-bound alpha-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing α-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 °C for 1 h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N) = 3) and 0.44 pmol (S/N = 10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1 g wet tissues were 3.67 μg (kidney), 1.97 μg (liver), 2.09 μg (heart), 0.59 μg (brain), 0.30 μg (lung), 0.38 μg (pancreas), and 0.20 μg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.     

高效液相色谱-柱后在线光化学衍生荧光检测法测定辣椒油中4种苏丹红染料

建立了在线光化学衍生、荧光检测、高效液相色谱(HPLC)测定辣椒油中苏丹红I、II、III和B的方法。以乙腈-水为流动相,采用梯度洗脱方式在SB-C18色谱柱上分离。用实验室自制的程序控制时间/光强光化学反应器作为在线衍生装置,优化了光衍生反应的条件和荧光检测条件。3种不同加标浓度下,辣椒油样品中4种苏丹红染料的加标回收率为81.3%～100.4%。加标水平为0.8 mg/kg下荧光信号强度的相对标准偏差(RSD,n=6)为2.6%～3.8%。苏丹红I、II、III和B的检出限(LOD)和定量限(LOQ)范围分别为0.009～0.054 mg/kg和0.030～0.181 mg/kg,优于传统的HPLC分离、二极管阵列检测器检测方法。该方法具有简单、灵敏、选择性好的特点,适用于食品样品中苏丹红的常规分析。     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

Determination of methoxsalen in dosage forms by high-performance liquid chromatography

Summary A new, rapid, sensitive, and specific method for the determination of methoxsalen in dosage forms using HPLC has been developed. methoxsalen is extracted in chloroform, evaporated on a water bath, and the residue is redissolved in ethanol. A standard solution of khellin (internal standard) in ethanol is added, and injected. A plot of peak height ratio (methoxsalen/internal standard) vs. concentration of methoxsalen gave a straight line (r=0.998). The column used was a stainless steel, 3.8 mm×30 cm, and the mobile phase was methanol: water (6040) at a flow rate of 2 cm³/min. Retention times for methoxsalen and khellin were 3.45 and 9.6 min, respectively. This method was found superior to the spectrophotometric assay in that no interference was encountered from structurally similar compounds or from coloring agents used in some commercial methoxsalen products.     

Computer-aided multichannel detection in high-performance liquid chromatography

Summary The development of a computer-aided rapid-scanning detector for HPLC based on the linear photodiode array UV-visible spectrophotometer is described. Chomatograms monitored at up to three wavelengths, with automatic capture of spectra for eluted peaks and post-run processing of spectral data to generate log₁₀ (A) spectra, second derivative and fourth derivative spectra, are described. Methods are reported for the analysis of forensic samples of diacetylmorphine (heroin) in the presence of the degradation products and potential contaminants caffeine, papaverine, 6-acetylcodeine, thebaine, 6-acetyl-morphine, procaine and morphine separated by HPLC. The novel use of second and higher derivative spectra for the qualitative characterisation of drugs and contaminants separated by HPLC is described.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of paralytic shellfish poisoning toxins in cultured microalgae by high-performance liquid chromatography with fluorescence detection

A novel method for the determination of paralytic shellfish poisoning (PSP) toxins using high-performance liquid chromatography with fluorescence detection was developed. The fluorescent derivates of neosaxitoxin (neoSTX), saxitoxin (STX), gonyautoxins 1 and 4 (GTX1+4), and gonyautoxins 2 and 3 (GTX2+3) were separated on a μBondapak NH₂ column (300 mm × 3.9 mm, 10 μm) using water and acetate buffer (pH 6.5) as the mobile phase (1.00 mL min⁻¹) in gradient mode with fluorescence detection at 390 nm (excitation at 330 nm). The linear ranges of neoSTX, STX, GTX1+4 and GTX2+3 were 3.31–331, 0.952–95.2, 3.78–378 and 0.124–12.4 ng mL⁻¹, respectively. The detection limits of neoSTX, STX, GTX1+4 and GTX2+3 were 1.10, 0.32, 1.26 and 0.041 ng mL⁻¹, respectively. The method was successfully applied to the determination of PSP toxins in microalgae. The recoveries ranged from 88±2% to 107±4% and the relative standard deviations were 0.16% to 4.4%. The procedure is also environmentally friendly because no organic solvent is used in the mobile phase.     

Determination of picomole amounts of carbonyls as luminarin hydrazones by high-performance liquid chromatography with fluorescence detection

Summary Fluorogenic reagents (luminarin 3, luminarin 11 and luminarin 12), having a quinolizinocoumarin moiety as fluorophore and a carboxylic acid hydrazide function as reacting group, have been developed. These reagents were found to be highly sensitive fluorescence derivatization reagents for aldehydes and ketones in high-performance liquid chromatography. The reagents readily react with carbonyl compounds in aqueous sulphuric acid solution (0.1 M) at room temperature to produce the corresponding hydrazone derivatives, which can be separated on both reversed or normal-phase column. The structures of the derivatives were studied, together with their properties in reversed and normalphase chromatographic systems. UV absorbance, corrected fluorescence spectral data and quantum yields of luminarin 3, luminarin 11 and luminarin 12 are presented. The detection limits (signal to noise ratio=3) for aldehydes and ketones were in the sub-pmol range. Luminarin 3 was also applied to the determination of hydroxymethylfurfural (HMF) in orange juices and concentrates. The method for HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to derivatization and normal-phase separation of the derivative with fluorimetric detection at 387 nmex., 444 nm em. The calibration curve was linear for amounts of HMF ranging from 0.1 to 10 nmol. Intrarun relative standard deviation was 12.8% for 0.1 nmol and 2.6% for 1 nmol. Recovery studies indicated an average of 98.7±1.9% for juice concentrate and 99.8±3.2% for pasteurized juice.