Determination of the nicotine metabolite trans-3'-hydroxycotinine in urine of smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring.

A gas chromatographic method for the determination of the nicotine metabolite trans-3'-hydroxycotinine is described. The method involves conversion of the metabolite to the tert.-butyldimethylsilyl derivative, chromatography on a fused-silica capillary column, and determination using nitrogen-phosphorus detection or electron ionization mass spectrometry with selected ion monitoring. A structural analogue, trans-3-hydroxy-1-methyl-5-(2-pyridyl)pyrrolidin-2-one (trans-3'-hydroxy-ortho-cotinine), was used as an internal standard. Using selected ion monitoring, good precision and accuracy were obtained for determination of trans-3'-hydroxycotinine in urine over the concentration range 10-10,000 ng/ml. There was a good correlation between concentrations determined by selected ion monitoring and by nitrogen-phosphorus detection in urine of smokers, although low concentrations determined using nitrogen-phosphorus detection tended to be somewhat higher, suggesting some interference from urinary constituents. Concentrations and 24-h excretion of trans-3'-hydroxycotinine in the urine of 22 cigarette smokers are reported and compared to concentrations and excretion of nicotine, cotinine, nicotine 1'-N-oxide, nornicotine, and cotinine N-oxide.     

Quantification of 2-hydroxyfluorene in human urine by column-switching high performance liquid chromatography with fluorescence detection

A high performance liquid chromatographic (HPLC) method for the quantification of 2-hydroxyfluorene (2-OHF) in normal human urine was established using deuterated 2-hydroxyfluorene (2-OHF-d9) as an internal standard with column-switching and fluorescence detection. The 2-OHF-d9 was synthesized by the metabolism of deuterated fluorene with cytochrome P450. The analytes were cleaned up on an ODS pre-column, via column-switching, and separated on an alkylamide-type reversed phase column. The internal standard eluted immediately prior to non-deuterated 2-OHF on the HPLC system and had nearly the same fluorescence characteristics as the non-deuterated 2-OHF. The detection limit was 0.03 nmol l(-1) (S/N = 3) and the calibration range of urine sample was from 0.2 to 50 nmol l(-1). The urine sample treatment involved enzymatic hydrolysis followed by solid phase extraction using a Sep-Pak C18 cartridge. 2-OHF was observed in the form of conjugates such as glucuronide and/or sulfate in human urine, and urinary metabolites were completely hydrolyzed for 2 h with beta-glucuronidase/aryl sulfatase. The proposed method was used to determine urinary 2-OHF in smokers and non-smokers, and showed that the urinary concentrations of 2-OHF in smokers were significantly higher than those in non-smokers (P < 0.01). Thus, the data suggest that urinary 2-OHF might be a sensitive and specific biological marker for the assessment of the exposure to polycyclic aromatic hydrocarbons.     

Exposure to 4,4'-methylenediphenyl diisocyanate (MDI) during moulding of rigid polyurethane foam: determination of airborne MDI and urinary 4,4'-methylenedianiline (MDA)

Occupational exposure to 4,4'-methylenediphenyl diisocyanate (MDI) was measured during moulding of rigid polyurethane foam. The aim of the study was to find out whether an MDI-derived urinary amine metabolite could be detected in the urine of workers exposed to apparently low levels of MDI. Airborne MDI was sampled on 1-(2-methoxyphenyl)-piperazine (2MP)-impregnated glass fibre filters and determined by high-performance liquid chromatography (HPLC) using ultraviolet (UV) and electrochemical (EC) detection. The limit of detection of MDI was 3 ng ml-1 for a 20 microliters injection. The precision of sample preparation, expressed as relative standard deviation (RSD), was 1.3% with UV detection and 2.1% with EC detection at a concentration of 70 ng MDI ml-1 (n = 6). The 2MP-MDI derivative was stable at +4 degrees C up to eight weeks. The accuracy of the method was validated in an international quality control programme. Workers (n = 57) from three different factories participated in the study. Urinary 4,4'-methylenedianiline (MDA) metabolite was determined after acid hydrolysis as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry using chemical ionisation and monitoring negative ions. The limit of detection in urine was 0.2 nmol l-1. The precision of six analyses for a urine sample spiked to a concentration of 1 nmol l-1 was 29% (RSD). The MDI concentrations were below the limit of detection in most (64%) of the air samples collected in the worker's breathing zone. Still, detectable amounts of MDA were found in 97% of the urine samples. Monitoring of urinary MDA appears to be an appropriate method of assessing MDI exposure in work environments with low or undetectable MDI concentrations in the workplace air.     

HPLC-based measurement of the chelator 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and its iron complex for pharmacokinetic studies in humans

An improved HPLC based method to assay the oral active iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, CP20) in serum and urine is described. The L1 peak has been well separated from other endogenous compounds, allowing the exact determination of the drug in both biological fluids. Moreover urinary iron excretion due to L1 therapy has been monitored by measuring urine Fe-(L1)₃ complex concentrations using reverse phase HPLC and subsequent detection at 450 nm. In patients and normal volunteers receiving this drug there is a good correlation between urine iron excretion measured by AAS and by the HPLC based method.     

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.     

Measurement of urinary estriol glucuronides during the menstrual cycle by high-performance liquid chromatography

A simple high-performance liquid chromatographic (HPLC) method for measuring estriol-3-glucuronide (E3-3-G) and estriol-16-glucuronide (E3-16-G) in the urine of non-pregnant women is described. Estriol conjugates were extracted from 4 ml of urine with a small cartridge of graphitized carbon black (Carbopack B). After washing, E3-3-G and E3-16-G were desorbed separately by a two-step elution system. After solvent removal, the two glucuronides were quantified by isocratic ion-suppression HPLC with fluorimetric detection. The analytical recovery of the two estriol metabolites was about 95%. The detection limit of the method was 0.6 ng/ml for both analytes in urine, which is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences. Data relative to urinary levels of E3-3-G and E3-16-G measured by this method on a daily basis in early morning samples from nine women during their menstrual cycles were compared with those reported in the literature and obtained by radioimmunoassay techniques. Moreover, the potential use of defined changes in the concentrations of the two conjugates for predicting the fertile period of women was assessed.     

Determination of 9-[(2-phosphonylmethoxy)ethyl]adenine in rat urine by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method for the determination of 9-[(2-phosphonylmethoxy)ethyl]adenine (PMEA) in urine is described. The procedure includes treatment of the urine sample with chloroacetaldehyde to form the fluorescent 1,N6-ethenoadenosine derivative, which was analyzed by reversed-phase HPLC with fluorometric detection. Validation of the method showed good sensitivity, precision and reproducibility. The method is useful for the study of urinary excretion of PMEA in the rat.     

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

Highly sensitive routine method for urinary 3-hydroxybenzo[a]pyrene quantitation using liquid chromatography-fluorescence detection and automated off-line solid phase extraction

Many workers and also the general population are exposed to polycyclic aromatic hydrocarbons (PAHs), and benzo[a]pyrene (BaP) was recently classified as carcinogenic for humans (group 1) by the International Agency for Research on Cancer. Biomonitoring of PAHs exposure is usually performed by urinary 1-hydroxypyrene (1-OHP) analysis. 1-OHP is a metabolite of pyrene, a non-carcinogenic PAH. In this work, we developed a very simple but highly sensitive analytical method of quantifying one urinary metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OHBaP), to evaluate carcinogenic PAHs exposure. After hydrolysis of 10 mL urine for two hours and concentration by automated off-line solid phase extraction, the sample was injected in a column-switching high-performance liquid chromatography fluorescence detection system. The limit of quantification was 0.2 pmol L(-1) (0.05 ng L(-1)) and the limit of detection was estimated at 0.07 pmol L(-1) (0.02 ng L(-1)). Linearity was established for 3-OHBaP concentrations ranging from 0.4 to 74.5 pmol L(-1) (0.1 to 20 ng L(-1)). Relative within-day standard deviation was less than 3% and relative between-day standard deviation was less than 4%. In non-occupationally exposed subjects, median concentrations for smokers compared with non-smokers were 3.5 times higher for 1-OHP (p<0.001) and 2 times higher for 3-OHBaP (p<0.05). The two urinary biomarkers were correlated in smokers (ρ=0.636; p<0.05; n=10) but not in non-smokers (ρ=0.09; p>0.05; n=21).     

Gas chromatographic-tandem mass spectrometric method for the quantitation of carbofuran, carbaryl and their main metabolites in applicators' urine

A new gas chromatographic-tandem mass spectrometric method has been developed and validated for the determination of two N-methylcarbamates, carbofuran and carbaryl and their metabolites in applicators' urine specimens. Mild conditions were used for sample preparation based on enzymic hydrolysis and solid-phase extraction using Oasis HLB sorbent cartridges. Amides, phenols and ketones were first converted to volatile derivatives of trifluoroacetic acid anhydride (TFAA) and afterwards were quantitated using tandem mass spectrometry. Linear calibration equations (1-200 ng mL(-1) urine) were obtained from fortified urine samples for all eight compounds, carbaryl, 1-naphthol, 2-naphthol, and carbofuran, 3-hydroxycarbofuran, 7-phenol, carbofuran-3-keto, 3- hydroxycarbofuranphenol. For all compounds, the limit of detection was lower than 0.1 ng mL(-1). Precision for all compounds, at the concentrations of 1, 10 and 100 ng mL(-1) (n = 5) in-fortified urine samples ranged from 0.7% to 18%. Accuracy was calculated at two concentrations 8 and 80 ng mL(-1) (n = 5) and ranged from -8.4% to 8.2%. Relative recoveries at concentrations of 1, 10 and 100 ng mL(-1), ranged from 71% to 116%. The method was successfully applied to five male applicators and 10 non-applicators (including both smokers and non-smokers).     

Method for measuring endogenous 3-O-methyldopa in urine and plasma.

The present report describes a method using column liquid chromatography with electrochemical detection for assaying concentrations of 3-O-methyldopa in urine and plasma. The technique combines a one-step sample preparation scheme with post-column flow-through electrodes in series, allowing adequate chromatographic separation of 3-O-methyldopa from other endogenous substances in urine. The validity of the method was confirmed by markedly decreased urinary 3-O-methyldopa levels after administration of an inhibitor of catechol-O-methyltransferase to rats, radioactivity in chromatographic fractions corresponding to 3-O-methyldopa in urine of rats undergoing infusion of [3H]-L-DOPA, and correlations between excretion rates of 3-O-methyldopa and catechols in humans. In healthy humans, urinary excretion of 3-O-methyldopa averaged 974 +/- 707 (S.D.) nmol per day, and plasma levels of 3-O-methyldopa averaged 89 +/- 32 nmol/l. The method should be useful in studies about the metabolism of endogenous and exogenous DOPA.     

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.     

Application of sample pre-oxidation of arsenite in human urine prior to speciation via on-line photo-oxidation with membrane hydride generation and ICP-MS detection

A pre-oxidation procedure which converts arsenite [As(III)] into arsenate [As(v)] was investigated in urinary arsenic speciation prior to on-line photo-oxidation hydride generation with ICP-MS detection. This sample pre-oxidation method eliminates As(III) and As(v) preservation concerns and simplifies the chromatographic separation. Four oxidants, Cl2, MnO2, H2O2 and I3-, were investigated. Chlorine (ClO-aq) and MnO2 selectively converted As(III) into As(v) in pure water samples, but the conversion was inefficient in the complex urine matrix. Oxidation of As(III) by H2O2 was least affected by the urine matrix, but the removal of excess H2O2 at pH 10 proved difficult. The most appropriate oxidant for the selective conversion of As(III) into As(v) with minimal interference from the urine matrix is I3- at pH 7. Unlike H2O2, excess oxidant can be easily removed by the addition of S2O3(2-). The I3-(-)S2O3(2-) treatment on a fortified sample of reconstituted NIST SRM 2670 freeze dried urine indicated that arsenobetaine (AsB), dimethlyarsinic acid (DMA), monomethylarsonic acid (MMA) and As(v) were not chemically degraded with recoveries ranging from 95 to 102% for all arsenicals. Sample clean-up involved pH adjustment prior to C18 filtration in order to achieve efficient As(III) conversion and quantitative recoveries of AsB and DMA. The concentrations determined in NIST SRM 2670 freeze dried urine were AsB 17.2 +/- 0.5, DMA 56 +/- 4 and MMA 10.3 +/- 0.3 with a combined total of 83 +/- 5 micrograms L-1 (+/- 2 sigma).     

Determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2h-1,4-benzodiazepin-2-one (Ro 7-9957) and its major biotransformation products in blood and urine by electron capture-gas-liquid chromatography.

A sensitive and specific electron capture-gas chromatographic assay was developed for the determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2H-1,4-benzodiazepin-2-one (I) and its major metabolites in blood and urine. The overall recovery of I and its N-desmethyl metabolite (II) from blood is apparently quantitative. The recovery of the major urinary metabolite, the N-desmethyl-3-hydroxy analog (IV), and the minor metabolites, the N-desmethyl analog (II) and the N-methyl-3-hydroxy analog (III) added to urine as authentic reference standards ranged from 80 to 85%. The sensitivity limits of detection are of the order of 2-3 ng of I and 4-5 ng of II per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion pattern in a dog following oral and intravenous administration of a 1-mg/kg dose (total 13 mg), and in man following the intravenous administration of single 5- and 10-mg doses. The N-desmethyl metabolite II was more predominant in dog blood than was the orally or intravenously administered I, but II was barely measurable in human blood.     

Simultaneous determination of perhexiline and its monohydroxy metabolites in biological fluids by gas chromatography-electron-capture detection

A rapid and sensitive method for the simultaneous determination of perhexiline and its cis-4-axial and trans-4-equatorial monohydroxy metabolites (M1 and M3, respectively) in human plasma, urine and bile is described. The assay utilises a single diethyl ether extraction, heptafluorobutyric acid anhydride derivatisation and separation and detection by gas chromatography-electron-capture detection. The limits of detection are 0.1 microgram/ml for perhexiline and 0.025 microgram/ml for the M1 and M3 metabolites. This method has been used in a five-day kinetic study of three healthy adult males who ingested a single 300-mg dose of perhexiline maleate. One of these volunteer subjects exhibited elevated plasma perhexiline and markedly reduced plasma and urinary M1 concentrations together with profoundly prolonged plasma and urinary M1 elimination times when compared with the other two subjects. These differences are thought to be of genetic origin. There were also obvious differences in urinary M3 concentrations which were discussed.     

A novel, selective, and rapid fluorimetric method for the simultaneous analysis of coproporphyrin and uroporphyrin in urine

A simple and rapid spectrofluorimetric method is described for the determination of the closely overlapping mixture of coproporphyrin (CP) and uroporphyrin (UP) in urine samples. Matrix Isopotential Synchronous Fluorescence Spectrometry (MISFS) was applied to improve the spectral resolution for the severely overlapped spectra of the urinary porphyrins. First-order derivative technique eliminates the background interference of each component on the other. Using these two techniques together, selectivity was improved, while maintaining a high sensitivity, and time-consuming separation processes and multiple scanning processes were avoided. The limits of detection were 0.15 nmol L⁻¹ and 0.1 nmol L⁻¹ for CP and UP, respectively. The concentrations of CP and UP were determined from the peak amplitudes of the Derivative Matrix Isopotential Synchronous Fluorescence (DMISF) spectra, at their detection points where the interference was suppressed. Porphyrins excretion in urine samples, collected from normal subjects, was studied. A comparison between the new method and the anion-exchange chromatographic method of Martinez and Mills was established using Bland-Altman method and the results indicate that these two methods are in a good agreement with each other.     

Quantitative determination of 8-isoprostaglandin F(2α) in human urine using microfluidic chip-based nano-liquid chromatography with on-chip sample enrichment and tandem mass spectrometry

Urinary 8-isoprostaglandin F(2α) (8-isoPGF(2α)) has been reported as an important biomarker to indicate the oxidative stress status in vivo. In order to quantitatively determine the low contents of 8-isoPGF(2α) (in sub- to low ng mL(-1) range) in physiological fluids, a sensitive detection method has become an important issue. In this study, we employed a microfluidic chip-based nano liquid chromatography (chip-nanoLC) with on-chip sample enrichment coupled to triple quadrupole mass spectrometer (QqQ-MS) for the quantitative determination of 8-isoPGF(2α) in human urine. This chip-nanoLC unit integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip. The introduction of enrichment column offers the advantages of online sample pre-concentration and reducing matrix influence on MS detection to improve sensitivity. In this study, the chip-nanoLC consisting of Zorbax 300A SB-C18 columns and Agilent QqQ Mass spectrometer were used for determining 8-isoPGF(2α) in human urine. Gradient elution was employed for effective LC separation and multiple reaction monitoring (MRM) was utilized for the quantitative determination of 8-isoPGF(2α) (m/z 353→193). We employed liquid-liquid extraction (LLE)/solid-phase extraction (SPE) for extracting analyte and reducing matrix effect from urine sample prior to chip-nanoLC/QqQ-MS analysis for determining urinary 8-isoPGF(2α). Good recoveries were found to be in the range of 83.0-85.3%. The linear range was 0.01-2 ng mL(-1) for urinary 8-isoPGF(2α). In addition, the proposed method showed good precision and accuracy for 8-isoPGF(2α) spiked synthetic urine samples. Intra-day and inter-day precisions were 1.8-5.0% and 4.3-5.8%, respectively. The method accuracy for intra-day and inter-day assays ranged from 99.3 to 99.9% and 99.4 to 99.7%, respectively. Due to its rapidity, enhanced sensitivity, and high recovery, this chip-nanoLC/QqQ-MS system was successfully utilized to determine the physiological biomarkers such as 8-isoPGF(2α) in human urine for clinical diagnosis.     

Enzymatic detection of urinary conjugated steroids after gel chromatography

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1-2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase--beta-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3 alpha-hydroxysteroid dehydrogenase or 3 beta-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3 beta-sulfates, and steroid-3 alpha-glucuronides and steroid-3 alpha-sulfates ae shown with some patients' samples. A precision of the assay values for steroid-3 alpha-glucuronide, steroid-3 alpha-sulfate and steroid-3 beta-sulfates in urine samples and assay values for normal subjects are also studied. This simple enzymatic method for detecting the excretion patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.     

磁固相萃取-高效液相色谱联用测定尿样中的1-羟基芘

采用磁固相萃取-高效液相色谱-荧光检测方法(MSPE-HPLC-FD)分析了尿样中芘代谢物1-羟基芘(1-Hydroxyperene 1-OHP).2mL尿样以0.1 mol/L醋酸钠溶液(pH 4.5)稀释至4 mL,酶水解后,再以0.1 mol/L醋酸钠溶液(pH 5.0)稀释至10mL,采用十八烷基膦酸改性的磁性介孔纳米粒子(50 mg)为萃取介质,对其进行MSPE富集,涡旋萃取1 min,甲醇解吸3min.解吸液经氮气吹干重新定容后,进行液相色谱分析.本方法在0.01～ 1.00 μg/L范围内线性良好(R2=0.9996);检出限为0.001μg/L日内相对标准偏差小于9.7％(n=5),日间相对标准偏差小于12.9％.将本方法应用于多个人体尿液样品中1-OHP含量的检测,结果满意.为确保结果的科学性和可靠性,测定结果用尿肌酐含量进行了归一化.