Aliquots Of Urine Samples Research Articles

Magnetic Solid Phase Extraction Followed by High Performance Liquid Chromatography for Determination of Urinary 1-Hydroxypyrene

A method was established for the determination of urinary pyrene metabolite 1-hydroxypyrene (1-OHP) by the combination of magnetic solid-phase extraction (MSPE) and liquid chromatography-fluorescence detection. Good extraction efficiency was obtained by optimizing the extraction conditions. A 2-mL aliquot of urine sample was diluted to 4 mL with sodium acetate (pH 4.5, 0.1 M) followed by enzymatic hydrolysis. The hydrolyzed sample was further diluted to 10 mL with sodium acetate (pH 5.0, 0.1 M) and then extracted by MSPE using n-octadecylphosphonic acid modified magnetic mesoporous nanoparticles (OPA/MMNPs, 50 mg). The extraction time and desorption time were 1 min and 3 min, respectively, with methanol as the desorption solvent. Desorption solution was evaporated to dryness under a mild nitrogen stream at 35 °C, and then dissolved in mobile phase for HPLC separation. The linearity range of the detection method was 0.01-1.0 μg L−1 with a correlation coefficient of 0.9996. The limit of detection was 0.001 μg L−1. The intra- and inter-day precisions (RSDs) were less than 9.7% (n = 5) and 12.9% (n = 3), respectively, which indicated this method has a satisfying reproducibility. The proposed method was further successfully applied to the determination of 1-OHP concentration in urine samples by normalizing to urinary creatinine. Collectively, the rapid, convenient and cost-effective method developed in current study offers a potential application to evaluate the human body PAHs exposure by determination of urinary 1-OHP.

A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry

The authors’ laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with β-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC ® BEH C18 column (10 cm L × 2.1 mm ID with 1.7 μm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors’ laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.

Influence of preservatives on the stability of ethyl glucuronide and ethyl sulphate in urine

Background Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. Methods Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4–8 °C), at room temperature (18 ± 1 °C) and in an incubator (36 ± 1 °C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC–ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. Results and conclusions Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC–MS/MS analysis.

Assessment of Smoking Status among Workers Using an Improved Colorimetric Method

To monitor smoking status among workers, we improved the colorimetric method to detect cotinine and other nicotine metabolites in urine. In adding ethanol in the reaction mixture, the quantitative measurable time defined as the duration with more than 95% of the peak absorbance, extended to 80 min in contrast to 16 min in the original method. As the analytical condition, a aliquot of urine sample (0.5 ml) was mixed with 0.5 ml of ethanol, with 0.2 ml of 4 M acetate buffer (pH: 4.7), with 0.1 ml of 150 mM KCN, with 0.1 ml of 0.44 M chloramin and then with 0.5 ml of 78 mM barbituric acid. A linear relationship was observed between cotinine concentration up to 80 microM and absorbance at 508 nm (r=0.998, p<0.01). Mean levels of nicotine metabolites among non smokers and smokers were 4.9 and 47.4 microM cotinine equivalent, respectively. Sensitivity and specificity were 96.1% and 96.7%, respectively for nicotine metabolites concentration among workers (n=385) when adopting 6.9 microM cotinine equivalent as a cut off value and the area under the ROC curve was 0.982. This method can be applicable to quantitative detection of smoking status.

The effect of grapefruit juice and seville orange juice on the pharmacokinetics of dextromethorphan: The role of gut CYP3A and P-glycoprotein

The objective of this study was to determine the effects of grapefruit juice and seville orange juice on dextromethorphan (DM) pharmacokinetics. Eleven healthy volunteers were studied over a 3-week period consisting of 5 study days each separated by a three-day washout. All subjects refrained from drinking caffeine containing beverages (coffee, soda, etc.) 8 h before orally taking DM (30 mg) with 200 ml water, 200 ml grapefruit juice, 200 ml water, 200 ml seville orange juice, and 200 ml water on Study Days 1 to 5. Aliquots of urine samples were assayed and analysed for DM, and the DM metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan using a validated HPLC method employing a phenyl column and a fluorescence detection. Results suggests that DM could provide some useful information on P-glycoprotein or related membrane efflux protein activity in the human gastro-intestinal tract. Bioavailability (F) of DM increased significantly with grapefruit and seville orange juice, but only returned to half the baseline value after three days of washout. This confirms that grapefruit and seville orange juice are long-lasting and perhaps irreversible inhibitors of gut CYP3A/P-glycoprotein. Grapefruit and seville orange juice appeared to have the same overall effect on DM pharmacokinetics. In addition, this paper presents a novel method of phenotyping for CYP2D6, CYP3A and P-glycoprotein using DM as a probe.

DETERMINATION OF STEROIDAL HORMONES IN URINE SAMPLES BY MICELLAR LIQUID CHROMATOGRAPHY FOLLOWING SOLID-PHASE EXTRACTION

Steroidal hormones were determined in spiked urine samples using micellar mobile phases of sodium dodecyl sulphate (SDS)-pentanol, solid-phase extraction (SPE), and detection in the UV region. In the optimized procedure, a 10 mL aliquot of urine sample is loaded into a C18 cartridge and washed with 2 mL of 50:50 (v/v) methanol-water, followed by 200 μL of 70:30 (v/v) methanol-water. The retained steroids are eluted with 2 mL of methanol and the eluate evaporated to dryness under nitrogen at 50°C. The residue is redissolved with 200 μL of the micellar mobile phase used in the chromatographic separation and injected into the chromatograph. The performance of the procedure was checked for 13 steroidal hormones: dehydrotestosterone, methandienone, methenolone enanthate, methyltestosterone, dydrogesterone, medroxyprogesterone, medroxyprogesterone acetate, nandrolone, nandrolone decanoate, progesterone, testosterone, testosterone enanthate, and testosterone propionate. Limits of detection were below 5 ng/mL for all steroids except nandrolone, nandrolone decanoate, and testosterone enanthate, whose elution from the SPE C18 column was not quantitative.

Development of a simple and rapid elisa of urinary cotinine for epidemiological application.

Highly stereospecific polyclonal antibodies (anti-CN) to cotinine (CN), a major metabolite of nicotine, were prepared from rabbit antisera to CN-linked keyhole limpet hemocyanin (KLH) by removing the antibodies to KLH and to its binding regions of CN. This was achieved by using immunoadsorbents consisting of insolubilized KLH onto CNBr-activated-Sepharose 4B. A new simple and rapid enzyme-linked immunosorbent assay (ELISA) of urinary CN was developed using the anti-CN. A brief outline of the method is as follows: CN-bovine thyroglobulin complex is coated onto wells of microtiter plates (1 ng/well), and then aliquots of urine samples or standard CN solutions were added followed by appropriate dilution of the anti-CN. The bound anti-CN antibodies are quantified spectrophotometrically with horseradish peroxidase-labelled anti-rabbit IgG and 2,2'-azino-di (3-ethylbenz- thiazoline)-6-sulfonic acid. Measurement of CN concentration in urine samples can be read off on a calibration curve drawn by using standard CN solutions. The standard curve ranged from 1 ng to 4 μ g /ml with an estimated lower limit of sensitivity of 7-8 ng/ml, resulting in within/between-assay CV (coefficient of variation) of lower than 10%. The method allowed one to assay more than 40 samples in duplicate by using just one plate, and is thus easily applicable to epidemiological investigations into exposure status to tobacco smoke.

Five treatment procedures evaluated for the elimination of ascorbate interference in the enzymatic determination of urinary oxalate

We evaluated the efficacies of five treatment procedures for eliminating ascorbate interference in the enzymatic determination of urinary oxalate. Aliquots of urine samples, containing different amounts of added ascorbate and oxalate, were individually subjected to ferric chloride, sodium nitrite, sodium periodate, charcoal, or ascorbate oxidase treatment to eliminate ascorbate interference. Oxalate contents of the urine samples were then determined by a banana oxalate oxidase-horseradish peroxidase-linked assay with 3-methyl-2-benzothiazolinone hydrazone and 3-(dimethylamino)benzoic acid as chromogens. Only those urine samples treated with ascorbate oxidase or charcoal consistently gave recovery of oxalate close to 100%. Treatment with other reagents, though improving the recovery of oxalate, gave inconsistent results. On the basis of these data, we describe procedures for simply and reliably assaying oxalate by using banana oxalate oxidase.