Fluorimetric high-performance liquid chromatography of prostaglandins and its application to their determination in human seminal fluid

A highly sensitive and simple high-performance liquid chromatographic method with fluorescence detection for the determination of prostaglandins is described. Prostaglandins are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 34 min (the total run time per injection, 56 min) on a reversed-phase column (YMC Pack C8) by a stepwise elution with mixtures of acetonitrile, methanol and water and detected fluorimetrically. The detection limits are 10-15 fmol at a signal-to-noise ratio of 5 in a 10-microliter injection volume. Prostaglandins E1, E2, F1 alpha and F2 alpha in human seminal fluids are measured by this method.     

高效液相色谱法测定大白鼠脊髓受到羟基游离基损伤时前列腺素的释放

研究了荧光衍生化试剂Ｂｒ－ＤＭＥＱ与前列腺素（ＰＧＦ２α）的反应条件及其荧光产物的色谱分离条件。方法灵敏度高,最小检测量为１０ｆｍｏｌ。并且利用微透析技术和Ｆｅｎｔｏｎ反应直接在脊髓内产生羟基游离基,测定了中枢神经系统受到羟基游离基损伤时ＰＧＦ２α的释放。     

High-performance liquid chromatography of arachidonic acid metabolites and its application to the determination of leukotriene B4 in stimulated leukocytes

A highly sensitive high-performance liquid chromatography with fluorescence detection for the determination of arachidonic acid metabolites is described. The metabolites are converted into corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-methylenedioxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Inertsil ODS) with aqueous acetonitrile and detected fluorimetrically. The detection limits are 5-15 fmol at a signal-to-noise ratio of 3 in a 10-microliter injection volume. The method is applied to the determination of leukotriene B4 produced in stimulated leukocytes.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Sensitive determination of bisphenol A and alkylphenols by high performance liquid chromatography with pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

A new and sensitive high-performance chromatographic method for the determination of bisphenol A and 8 alkylphenols with fluorescence detection is reported. Each phenol was derivatized by reaction with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole at 40 degrees C for 60 min. The fluorescence derivatives were separated on a Wakosil 5C18 column (4.0 i.d. x 300 mm, 5 microm) with methanol:water (10:90) as mobile phase (detection wavelength: lambda(ex) 336 nm, lambda(em) 440 nm). The detection limits were in the range of 0.1-10.0 pg/mL in serum. The calibration graphs were linear to 1.0 microg/mL. The relative standard deviations were 7.2-8.9%, respectively. The proposed method was applied to the determination of bisphenol A in mother and infant rat serum.     

Diethylhydrogensilyl-cyclic diethylsilylene derivatives in gas chromatography-mass spectrometry of prostanoids. III. F alpha-prostaglandins and thromboxane B2 derivatives

The gas chromatographic (GC) and GC-mass spectrometric properties of the diethylhydrogensilyl-cyclic diethylsilylene (DEHS-DES) derivatives of prostaglandin (PG) F1 alpha methyl ester, PGF2 alpha methyl ester, 6-keto-PGF1 alpha methyl ester-alkyloxime and thromboxane (TX) B2 methyl ester-alkyloxime and the DES derivative of 13,14-dihydro-15-keto-PGF2 alpha methyl ester-alkyloxime were studied. When the ketonic PGs and TXB2 were converted into their methyloxime derivatives, the methylene unit values of these five prostanoid derivatives were slightly greater than those of the corresponding dimethylethylsilyl ether derivatives. When the ketonic PGs were converted into their corresponding ethyloxime derivatives, baseline separation was achieved in 20 min by use of a methylsilicone cross-linked fused-silica capillary column. The mass spectra of these derivatives were characterized by the ion at m/z 157 for F alpha prostaglandins and m/z 269 for TXB2. The major fragmentations were directed by the DES group, and other fragmentations common to the prostanoid derivatives were losses of an ethyl radical at the silicon atom, C5H11 hydrocarbon fragment, diethylhydrogensilanol and C15-C20 hydrocarbon fragment. The mass fragmentations of these prostanoid derivatives are briefly discussed. GC with high-resolution selected-ion monitoring was carried out for the TXB2 derivative at a resolution of 8000 by monitoring the ion at m/z 269.1573. A 25-pg amount of this derivative showed a well shaped doublet with a signal-to-noise ratio of more than 300:1.     

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.     

胆汁酸的双敏感探针苯并[b]吖啶酮-5-乙基对甲苯磺酸酯:荧光检测和APCI-MS鉴定

在HypersilC18色谱柱上,利用新型荧光试剂苯并[b]吖啶酮-5-乙基对甲苯磺酸酯(BAETS)作柱前衍生化试剂,采用梯度洗脱对10种胆汁酸衍生物进行了优化分离.在二甲亚砜溶剂中,以碳酸钾作催化剂,95℃,45min后获得稳定的荧光产物.衍生物在稳态荧光条件下,在乙腈和甲醇水溶液中的百分离子化δ值在0%～88.83%和0%～89.15%范围内.最大激发和发射波长为λex/λem=272/505nm.采用大气压化学电离源(APCI)正离子模式实现了胆汁中胆汁酸的定性定量测定.线性回归系数均在0.9995以上,线性范围宽,检出限为0.76～1.62ng/mL.     

(10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3: an improved synthesis via 19-nor-10-oxo-vitamin D

An efficient synthetic route to (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 was developed. The key feature of this pathway is the introduction of a 19-fluoromethylene group to a (5E)-19-nor-10-oxo-vitamin D derivative. The 10-oxo-compound was obtained via a 1,3-dipolar cycloaddition reaction of (5E)-1alpha,25-dihydroxyvitamin D with in situ generated nitrile oxide followed by ring cleavage of the formed isoxazoline moiety with molybdenum hexacarbonyl. Conversion of the keto group of (5E)-19-nor-10-oxo-vitamin D to the E and Z fluoromethylene group was achieved through a two-step sequence involving a reaction of lithiofluoromethyl phenyl sulfone followed by the reductive desulfonylation of the alpha-fluoro-beta-hydroxy sulfone. The dye-sensitized photoisomerization of the (5E)-19-fluorovitamin D afforded the desired (5Z)-19-fluorovitamin D derivatives, (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3.     

Determination of 21-hydroxycorticosteroids in human urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method with fluorescence detection for the determination of nineteen 21-hydroxycorticosteroids is described. The corticosteroids are oxidized by cupric acetate to form the corresponding glyoxal derivatives. The derivatives are converted into fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-dicarbonyl compounds. The quinoxalines are separated within 70 min on a reversed-phase column (TSK gel ODS-120T) by stepwise elution with mixtures of methanol, acetonitrile, and 1.0 M ammonium acetate. The detection limits are 0.14-29.4 pmol at a signal-to-noise ratio of 3 in a 50-microliter injection volume. This sensitivity permits precise determination of hydrocortisone, cortisone, corticosterone, and their tetrahydro derivatives in 500 microliters of normal human urine.     

Development of 7-(N,N-dimethylaminosulfonyl)-5-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole as a water-soluble fluorogenic reagent for the sensitive liquid chromatographic determination of saturated carboxylic acids

A reversed-phase high-performance liquid chromatographic (HPLC) method for the femtomole determination of nine saturated carboxylic acids, n-butyric (C4), n-hexanoic (C6), n-caprylic (C8), n-decanoic (C10), lauric (C12), n-tetradecanoic (C14), palmitic (C16), stearic (C18) and arachidic (C20), based on the condensation reaction of these acids with a newly synthesized water-soluble benzofurazan fluorescent reagent, 7-(N,N-dimethylaminosulfonyl)-4-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH2), was developed. The derivatization reaction proceeds with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) in the presence of the catalyst 4-(dimethylamino)pyridine (DMAP). A model derivative of the reagent with n-caprylic acid (C8) was synthesized for fluorescence excitation/emission characterization. Depending on the solvents, including water, methanol, acetonitrile, 1,4-dioxane or N.N-dimethyformamide (DMF), the C8 derivative has a fluorescence emission with a fluorescence quantum yield (phi) ranging from 0.01 to 0.20 in the region from 545 to 580 nm. An exponential increase in phi was observed with increasing acetonitrile content. The calculated detection limits (signal-to-noise ratio = 3:1) of the proposed method for the above nine carboxylic acids were 9.1, 4.0, 2.5, 2.2, 2.0, 1.8, 1.2, 1.0 and 1.3 fmol, respectively. Biological samples including Intralipos 20% and rat plasma were analysed satisfactorily.     

5-nitro-5-(1′-cyclohexenyl)- and 5-nitro-5-(1′-cycloheptenyl)-3-benzyl- and 3-cyclohexyl tetrahydro-1,3-oxazines

1-(Cyclohexenyl- and 1-cycloheptenylnitromethane have been used as starting substances to yield 2-nitro-2-(1′-cyclohexenyl)- and 2-nitro-2-(1′-cycloheptenyl)-propanediol-1,3 (B) respectively. By reacting them with formaldehyde and benzylamine or formaldehydes and cyclohexylamine, derivatives of tetrahydro-1,3-oxazine (B) have been prepared. Acid hydrolysis of tetrahydro-1,3-oxazine derivatives results in the formation of 3-hydroxy-2-nitro-2-(1′-cyclohexenyl)- and 3-hydroxy-2-nitro-2-(1′-cycloheptenyl)-propylamine derivatives (E). Both aminoalcohols (E) react with formaldehyde to again yield tetrahydro-1,3-oxazine derivatives (C).

Infra-red spectra of C and E and their hydrochlorides D and F were examined and structure assignments made of the principal bands.     

反相高效液相色谱法分离2-(氟苯基)-5-甲基苯并恶唑和2-(氯苯基)-5-甲基苯并恶唑的位置异构体

基于商品化的普通色谱柱建立了2-(氟苯基)-5-甲基苯并恶唑和2-(氯苯基)-5-甲基苯并恶唑邻、间、对位置异构体的分离检测方法。色谱柱为Inertsil ODS-SP C₁₈(250 mm×4.6 mm, 5 μm),以乙腈(A)和水(B)为流动相,在60%A~80%A间线性梯度洗脱15 min,流速为1.5 mL/min,柱温40℃,检测波长为310 nm。在质量浓度为2~200 mg/L范围内,2-(氟苯基)-5-甲基苯并恶唑邻、间、对位的异构体、2-(氯苯基)-5-甲基苯并恶唑邻、间、对位的异构体具有良好的线性关系,6种化合物的检出限(S/N=3)依次为0.0307、0.0293、0.0315、0.0226、0.0237、0.0226 mg/L。该法既为5-甲基苯并恶唑与氟苯或氯苯碳氢活化偶联反应制备的异构体混合物提供了一个快速检测的方法,又为2-芳基苯并恶唑类异构体的分离检测提供了参考。     

Rapid and ultrasensitive determination of ephedrine and pseudoephedrine derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper presents a micellar electrokinetic chromatography method with laser-induced fluorescence detection to analyze ephedrine (E) and pseudoephedrine (PE) after derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein. The optimum derivatization conditions were: 0.05 M Na2CO(3/NaHCO3 (pH 9.5), reaction time 30 min at 45 degrees C, molar ratio of DTAF to E and PE mixture 20:1. The baseline separation was achieved within 8 min with running buffer composed of 20 mM borate+20 mM SDS+15% acetonitrile (v/v) (adjusted pH 9.8), and applied voltage of 20 kV. Good linearity relationships (correlation coefficients: 0.9906 for E and 0.9941 for PE) between the peak heights and concentration of the analytes were obtained (2.5-50 ngmL(-1)). The detection limits for E and PE were 3.85 x 10(-4) and 1.41 x 10(-4)ngmL(-1), respectively, which indicated that the proposed method surpassed other chromatographic alternatives in terms of limit of detection by at least 10(3) folds. The method was applied to the analysis of the two alkaloids in ephedra herb plants and its preparations with recoveries in the range of 89.6-107.0%.     

Sensitive determination of aliphatic amines by high-performance liquid chromatography with a new fluorogenic probe 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde

A new fluorogenic reagent 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde (FBQCA) has been synthesized and used as a derivatizing reagent for the determination of aliphatic amines with HPLC. The reagent is nonfluorescent, but forms highly fluorescent isoindole upon the reaction with primary amines in alkaline medium. Eleven amine derivatives were baseline separated in 8 min using a gradient elution on a C(8) column and detected with fluorescence detection at lambda(ex)/lambda(em) = 480/546 nm. The detection limits were in the range of 0.5-2 nM (S/N = 3). The proposed method has been successfully applied to the analysis of aliphatic amines in food and environmental samples, including white wine, soybean oil, soil, and tap water with satisfactory recoveries in the range of 94-106%.     

Microemulsion electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive microemulsion electrokinetic chromatography with laser-induced fluorescence detection method was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. By a series of optimization, a running buffer composed of 20 mM borate + microemulsion (23.3 mM Sodium dodecyl sulfate/180.85 mM 1-butanol/16.4 mM n-heptane) +8% acetonitrile was applied for the separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.058-11.58 microg.mL(-1) (correlation coefficient: 0.9993 for E, 0.9995 for PE), and the detection limits for E and PE were 5.3 and 3.9 ng.mL(-1). The method was applied to the analysis of the two alkaloids in Chinese traditional herbal preparations with recoveries in the range of 96.9-105.4%.     

Dual-sensitive probe 1-imidazole-2-(5-benzoacridine)-ethanone for the determination of amines in environmental water using HPLC with fluorescence detection and online atmospheric chemical ionization-mass spectrometry identification

Dual-sensitive probe of 1-imidazole-2-(5-benzoacridine)-ethanone (IBAE) for the determination of free amines with fluorescence detection and online highly sensitive atmospheric chemical ionization-mass spectrometry identification (APCI-MS) has been developed. 2-(Benzoacridine)-5-acetic acid (BAAA) reacts with coupling agent N,N′-carbonyldiimidazole (CDI) to form a highly activated amide intermediate 1-imidazole-2-(5-benzoacridine)-ethanone (IBAE), which is dual-sensitive probe. The amide intermediate (IBAE) reacts preferably with amines in dimethylformamide (DMF) solvent to give the high yields of derivatives. IBAE-amine derivatives are not only sensitive to fluorescence but also to MS ionizable efficiency. The percent ionization δ values change from 0 to 57.32% in aqueous acetonitrile and from 0 to 62.14% in aqueous methanol. The relative standard deviations of the peak areas with fluorescence detection for each amine are <1.24% (40 ng/ml, n = 6). The fluorescence detection limits (at a signal-to-noise ratio of 3) are in the range of 0.15-0.50 ng/ml. The online APCI-MS detection limits are in the range of 2.07-8.51 ng/ml (at a signal-to-noise ratio of 3). Therefore, the facile IBAE intermediate derivatization allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amines in environmental water.     

Vinyltriphenylphosphonium Salt Mediated One-Pot Stereoselective Synthesis of Dialkyl ( E )-2-(2-methyl-5-oxo-1-cyclopentenyl)-2-butenedioates

Protonation of the highly reactive 1:1 intermediates produced in the reaction between triphenylphosphine and dialkyl acetylenedicarboxylates by 2-hydroxy-3-methyl-2-cyclopenten-1-one leads to vinyltriphenylphosphonium salts, which undergo an intramolecular Wittig reaction to produce the corresponding cyclobutene derivatives. The cyclobutene derivatives are not isolable and undergo electrocyclic ring-opening reactions in CH 2 Cl 2 at room temperature to produce dialkyl ( E )-2-(2-methyl-5-oxo-1-cyclopentenyl)-2-butenedioates in moderate yields.     

Reaction of chloromethyl derivatives of ethyl 3-(furyl)-3-(diethoxyphosphoryl)acrylates with sodium azide and potassium thiocyanate

Reaction of chloromethyl derivatives of ethyl 3-furyl-3(diethoxyphosphoryl)acrylates with sodium azide in acetonitrile in the presence of catalytic amount of potassium iodide proceeds with substitution of halogen with the azido group. The same chloromethyl derivatives react with potassium thiocyanate under analogous conditions to give a mixture of thiocyanates and isothiocyanates in (0.6–0.8) : 1 ratio save the case of ethyl 3-(3-chloromethylfur-2-yl)- and 3-(4-chloromethylfur-3-yl)acrylates when only thiocyanates are formed. Bromination of diethyl 5-methyl-2-furoyl phosphonate with N-bromosuccinimide afforded 5-bromomethyl-2-furoyl phosphonate. In the reaction with potassium thiocyanate it forms only thiocyanate.     

胆汁酸的双敏感探针苯并[b]吖啶酮-5-乙基对甲苯磺酸酯: 荧光检测和APCI-MS鉴定

在Hypersil C₁₈色谱柱上, 利用新型荧光试剂苯并[b]吖啶酮-5-乙基对甲苯磺酸酯(BAETS)作柱前衍生化试剂, 采用梯度洗脱对10种胆汁酸衍生物进行了优化分离. 在二甲亚砜溶剂中, 以碳酸钾作催化剂, 95 ℃, 45 min后获得稳定的荧光产物. 衍生物在稳态荧光条件下, 在乙腈和甲醇水溶液中的百分离子化δ值在0%～88.83%和0%～89.15%范围内. 最大激发和发射波长为λ_ex/λ_em＝272/505 nm. 采用大气压化学电离源(APCI)正离子模式实现了胆汁中胆汁酸的定性定量测定. 线性回归系数均在0.9995以上, 线性范围宽, 检出限为0.76～1.62 ng/mL.