Nonionic surfactant‐capped gold nanoparticles for selective enrichment of aminothiols prior to CE with UV absorption detection

In this study, we describe the use of Tween 20‐capped gold nanoparticles (AuNPs) as selective probes for the extraction of aminothiols from an aqueous solution. Tween 20 molecules noncovalently attached to the surface of AuNPs to form Tween 20–AuNPs were used for the selective extraction of aminothiols through the formation of Au–S bonds. After extraction and centrifugation, the aminothiols were detached from the surface of the AuNPs by adding DTT in a high concentration. We used this probe in combination with CE and UV absorption detection. On‐line concentration and separation of the released aminothiols were performed by using 1.6% v/v poly(diallyldimethylammonium chloride) as an additive in CE. Under optimal extraction and stacking conditions, the LOD at a S/N of 3 were 28, 554, and 456 nM for glutathione (GSH), cysteine (Cys), and homocysteine (HCys), respectively. In comparison with the normal injection without the extraction procedure, approximately 2280‐, 998‐, and 904‐fold improvements in the sensitivity were observed for GSH, Cys, and HCys, respectively. We have validated the application of our method on the basis of the analysis of GSH and HCys in human urine samples. It is believed that this approach has significant potential to be extended to clinical diagnosis.     

金纳米粒子富集-高效液相色谱同时测定人血浆中三种氨基硫醇

建立了金纳米粒子富集-高效液相色谱-紫外检测(HPLC-UVD)同时测定人血浆中3种氨基硫醇(半胱氨酸(Cys)、高半胱氨酸(Hcys)、谷胱甘肽(GSH))的新方法。以Tween 20修饰的金纳米粒子作为选择探针萃取富集氨基硫醇。经二硫苏糖醇脱附后,采用SpursilTM C18柱(250 mm×4.6 mm, 5 μm)分离氨基硫醇,以60 mmol/L磷酸盐缓冲溶液(pH 2.0)等度洗脱,检测波长为200 nm。3种氨基硫醇的浓度分别在0.025～350 μmol/L、0.02～60 μmol/L、0.01～50 μmol/L内与峰面积具有良好的线性关系,相关系数均高于0.99。方法检出限(信噪比为3)分别为5.0、6.0和2.5 nmol/L,回收率为92.8%～106.0%。该方法能显著降低血浆样品中内源性物质的干扰,提高HPLC-UVD的选择性和灵敏度。将该方法应用于心血管病人血浆中上述氨基硫醇的分离测定,结果显示: 与对照组相比,疾病组血浆中的Hcys和GSH水平存在显著性差异,Cys不存在显著性差异。     

Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1–12 nmol L⁻¹ for standard solution (high femotomole, absolute), except AdoMet (116 nmol L⁻¹), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4 h.     

Simultaneous analysis of plasma thiols by high-performance liquid chromatography with fluorescence detection using a new probe, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene

The design, synthesis and properties of a new derivatizing reagent, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene (TMPAB-I), for thiol groups are presented. Using the derivatization of TMPAB-I with thiols, a new high-performance liquid chromatographic method for measuring low-molecular-weight thiol-containing compounds, including coenzyme A (CoA), glutathione, N-acetylcysteine, cysteine, homocysteine (HCys) and 6-mercaptopurine has been developed. The reaction of TMPAB-I with thiols is specific, fast and stable for both TMPAB-I and the derivatives. A baseline separation of all the six derivatives is achieved by isocratic elution on reversed-phase column within 20 min with detection wavelengths of 500 and 510 nm for the excitation and emission, respectively, and the limits of detection (signal-to-noise ratio = 3) are from 1.8 fmol (CoA) to 14.0 fmol (HCys), respectively, per 20 μL injection. The utility of the proposed method has been validated by measuring thiol-containing compounds in human plasma samples from healthy persons and patients with hypertension, with recoveries of 94.2–106.8%.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

Sodium hydroxide as pretreatment and fluorosurfactant-capped gold nanoparticles as sensor for the highly selective detection of cysteine

A sensor for detecting cysteine (Cys) in a solution of fluorosurfactant (FSN)-capped gold nanoparticles (AuNPs) has been developed. Under acidic conditions, FSN-capped AuNPs are aggregated in the presence of homocysteine (HCys) and Cys but not in the presence of cysteinylglycine, glutathione, and gamma-glutamycysteine. When adding NaOH to a solution of HCys, the five-membered ring transition state is formed through intramolecular hydrogen abstraction. By contrast, it is difficult for Cys to form a four-membered ring transition state after Cys has been pretreated with NaOH. As a result, the HCys-induced aggregation of the FSN-capped AuNPs is suppressed because the five-membered ring transition state exhibits relatively larger steric hindrance and has stronger interaction with the FSN molecules. Thus, we can discriminate between Cys and HCys on the basis of different aggregation kinetics. Under the optimum condition, the selectivity of the probe for Cys in aqueous solutions is remarkably high over the other aminthiols. Note that HCys and Cys have very similar structure and pK(a) value. We have validated the applicability of our method through the analyses of Cys in urine samples. It is believed that this approach has great potential for the detection of Cys in biological samples.     

Second-order calibration of excitation-emission matrix fluorescence spectra for determination of glutathione in human plasma

A rapid non-separative spectroflourimetric method based on the second-order calibration of the excitation-emission data matrix was proposed for the determination of glutathione (GSH) in human plasma. In the phosphate buffer solution of pH 8.0 GSH reacts with ortho-phthaldehyde (OPA) to yield a fluorescent adduct with maximum fluorescence intensity at about 420 nm. To handle the interfering effects of the OPA adducts with aminothiols other than GSH in plasma as well as intrinsic fluorescence of human plasma, parallel factor (PARAFAC) analysis as an efficient three-way calibration method was employed. In addition, to model the indirect interfering effect of the plasma matrix, PARAFAC was coupled with standard addition method. The two-component PARAFAC modeling of the excitation-emission matrix fluorescence spectra accurately resolved the excitation and emission spectra of GSH, plasma (or plasma constituents). The concentration-related PARAFAC score of GSH represented a linear correlation with the concentration of added GSH, similar to that is obtained in simple standard addition method. Using this standard addition curve, the GSH level in plasma was found to be 6.10 ± 1.37 μmol L⁻¹. The accuracy of the method was investigated by analysis of the plasma samples spiked with 1.0 μmol L⁻¹ of GSH and a recovery of 97.5% was obtained.     

Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection

A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as l-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-β-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ = 410 nm). With the optimized conditions for chromatography and the post-column derivatization, 40 nM of NAC, 40 nM of Cys, and 50 nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2–5.4% for 50 μM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410 nm (in the range of 0.04–0.58 μM) after post-column derivatization with DTNB + M-β-CD were much lower than those at 205 nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.     

Preparation of gold nanoparticles on eggshell membrane and their biosensing application

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25 ± 7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl₄ without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl₄) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH ≤3.0 and ≥7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO_x) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO_x. The linear response range of the glucose biosensor is 20 μM to 0.80 mM glucose with a detection limit of 17 μM (S/N = 3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.     

Kinetic spectrophotometric determination of Tween 80

A novel kinetic spectrophotometic method for the determination of Tween 80 based on its interaction with 5(p-dimethylaminobenzylidene)rhodanine (PDR) in alkaline media is reported. The effect of variable on the rate of interaction of Tween 80 and PDR was investigated in order to establish the optimum conditions. The interaction was monitored spectrophotometically and change in absorbance (ΔA) of PDR at 464 nm at times of 30 and 270 s was used as an analytical parameter. Tween 80 can be measured in the range of 2.5×10⁻⁵ to 1.25×10⁻³ M with detection limit of 1.5×10⁻⁵ M. The relative standard deviation for eight replicate determinations of 2×10⁻⁴ and 1×10⁻³ M of Tween 80 solution was 4.08 and 3.88%, respectively. This method was used to determine Tween 80 in biscuit and multivitamin syrup.     

HPLC-electrospray tandem mass spectrometry for simultaneous quantitation of eight plasma aminothiols: Application to studies of diabetic nephropathy

It was reported that Hcy was related to the development of kidney disease, but it remains unknown whether Hcy is an independent biomarker for diabetic nephropathy. Analytical method for simultaneous determination of aminothiols among the Hcy metabolic cycle is desirable to discover other potential biomarkers. A high-performance liquid chromatography-electrospray tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for simultaneous quantitation of Cysteine (Cys), total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine (Cysta), methionine (Met), glutathione (GSH) and cysteinylglycine (Cys-gly) in plasma with N-(2-mercaptopropionyl)-glycine (MPG) as internal standard. The method had simple pretreatment without derivatization and the chromatograms show better separation of the eight aminothiols and the analytic time was 20 min. The results demonstrated that it provided an excellent linearity for all analytes over their respective concentration ranges and illustrated excellent precision and plasma recovery as well. Then, the method was applied in the case-control study of patients with diabetes mellitus (DM) and diabetic nephropathy (DN). In conclusion, it is an effective method to quantitate the concentrations of aminothiols in the human plasma. SAH and SAM were suggested as better potential biomarkers of DM and DN.     

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: Application to the rapid screening of 5S rRNA from ovarian cancer cells

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (M_avg = 4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL⁻¹) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL⁻¹ with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps.     

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λ_ext/λ_em = 340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L⁻¹ CH₃COONH₄ (pH = 2.5)/CH₃CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25–5.0 μmol L⁻¹ and the LOD was 19 nmol L⁻¹. No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.     

A potential visual fluorescence probe for ultratrace arsenic (III) detection by using glutathione-capped CdTe quantum dots

The interaction between mercaptoacetic acid (MA)-capped CdTe QDs, MA-capped CdTe/ZnS QDs or glutathione (GSH)-capped CdTe QDs with As(III) was studied using fluorescence spectrometry. As (III) has a high-affinity to reduced-GSH to form As(SG)₃, and the emission of the GSH-capped CdTe QDs (λ_em. = 612 nm) is quenched effectively. Thus, a novel fluorescence spectrometric method was developed for As (III) determination by using GSH-CdTe QDs. Under optimal conditions, the quenched fluorescence intensity (F₀/F) increased linearly with the concentration of As (III) ranging from 5.0 × 10⁻⁶ to 25 × 10⁻⁵ mol L⁻¹. The limit of detection (3σ) for As (III) was found to be 2 × 10⁻⁸ mol L⁻¹. This method is potentially useful in visual detection of As (III) under irradiation of the ultraviolet light.     

A novel electrochemical sensing strategy for rapid and ultrasensitive detection of Salmonella by rolling circle amplification and DNA–AuNPs probe

A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA–AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10 aM to 10 pM with a detection limit of 6.76 aM (S/N = 3). The developed method had been successfully applied to detect Salmonella as low as 6 CFU mL⁻¹ in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring.     

Inner filter effect of gold nanoparticles on the fluorescence of quantum dots and its application to biological aminothiols detection

We have demonstrated the design of a new type fluorescence assay based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of quantum dots (QDs). With a high extinction coefficient, AuNPs are expected to be capable of functioning as powerful absorbers. QDs with tunable emission wavelength are ideal fluorophores because the emission spectra of the rationally synthesized QDs can perfectly overlap with the absorption band of the absorber. Aminothiols are chosen as the model analytes, and the IFE-based fluorescent method for detection of aminothiols was suggested. Under the optimum conditions, the response is linearly proportional to the concentration of cysteine in the range of 0.05-0.9 μg mL⁻¹. The present IFE-based fluorescent strategy could be also used to detect glutathione and homocysteine. The linear concentration ranges were 0.05-1.0 μg mL⁻¹ for glutathione and 0.01-1.0 μg mL⁻¹ for homocysteine.     

Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.     

Determination of aminothiols by liquid chromatography with amperometric detection at a silver electrode: Application to white wines

Liquid chromatography coupled to a silver electrode based flow-through amperometric detector (LC-EC-Ag) was developed for the determination of aminothiols in white wines. The C18 reversed phase LC system operated in the isocratic mode at 0.7 mL min⁻¹ and used an acidic mobile phase composed of formic acid, EDTA, sodium nitrate, sodium hydroxide, and methanol 1% (v/v) at pH 4.5. The working electrode operated at 0.08 V vs Ag/AgCl, 3 M KCl and its manual cleaning was realized once a month by smoothing on a polishing cloth. The analyzed aminothiols were resolved and eluted within 4 min, and all standard curves were linear in the range 2 × 10⁻⁷–2 × 10⁻⁵ M. The analyzed wine samples needed no preparation other than dilution with the mobile phase. The concentration of cysteine (CYS), homocysteine (HCYS), glutathione (GSH) and N-acetylcysteine (NAC) in bottled white wines, determined by the method of standard addition, was found to be in the low μM range (0.2–2 mg L⁻¹) depending on the wine type and its age.     

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

Determination of 6-mercaptopurine based on the fluorescence enhancement of Au nanoparticles

A novel method for the determination of 6-mercaptopurine (6MP) has been developed based on fluorescence enhancement of Au nanoparticles (AuNPs). The fluorescent AuNPs with mean diameter of ∼15 nm were synthesized in aqueous solution, exhibiting the stable maximum emission at 367 nm, under the excitation at wavelength of 264 nm. The AuNPs self-assembly with 6MP were characterized with transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) absorption, fluorescence and surface-enhanced Raman scattering (SERS) spectroscopy. The results revealed that the surface attachment through versatile binding sites of S10, N3, N9 and N7 atoms in 6MP produced the interparticle coupling and formed aggregates of AuNPs. As a result, the fluorescence emission enhancement was significantly observed upon AuNPs self-assembly with 6MP. The fluorimetric determination under optimal conditions indicated that 6MP could be quantified in good linearity range of 6.35 × 10⁻⁸ to 3.05 × 10⁻⁷ M, with a low detection limit of 4.82 × 10⁻¹⁰ M. The relative standard deviation (n = 11) was 1.8% at 2.54 × 10⁻⁸ M 6MP concentration level. The proposed method was successfully applied for the determination of 6MP in spiked human urine. The probable fluorescence enhancement mechanism was also discussed there.