Freeze-dried Human Urine Research Articles

Thiol-functionalized MOF modified 3D printed monolithic microextraction array for analysis of trace Cd and Pb in human urine

Controlling the position, size, and shape of pores is a limitation of traditional monolithic preparation methods. The application of 3D printing technology offers high customizability, allowing the precise printing of pore positions, sizes, and shapes according to the designer's 3D model. Herein, by using Projection Microstereolithography (PμSL), we prepared a 3D-printed monolithic array with post-modification of thiol-functionalized metal-organic framework (MOF), and combined it with inductively coupled plasma mass spectrometry (ICP-MS) for the online analysis of trace Cd and Pb in human urine. To achieve array monolithic microextraction, six 3D-printed monolithic columns were modified with thiol-functionalized MOF-808 (MOF-808-SH), and were then assembled in the 3D printed extraction device incorporating gas valve and scaffold. The MOF-808-SH modified 3D-printed monolithic column exhibits excellent extraction performance to Cd2+ and Pb2+ due to rich active adsorption sites and hierarchical porous structure, and has long life span (>100 reused times). Under the optimized conditions, the limits of detection (LODs) are 3.5 and 17.6 ng L−1 for Cd2+ and Pb2+, respectively, with the relative standard deviations of 4.9 % and 8.2 % (0.1 μg L−1, n = 7), and the sample throughput is 11 h−1. To validate the accuracy of the method, the method was used to determine Cd and Pb in Certified Reference Materials of freeze-dried human urine, the determined results agree well with the certified values. This method was also successfully applied to the determination of trace Cd and Pb in real human urine samples. The developed method offers low LODs, robust anti-interference capability, high sample throughput, long reuse cycles, and automation analysis, showing great potential for the analysis of trace heavy metals in biological samples.

Development of new matrix reference materials for quantitative urine analysis in drug tests.

Accurate quantitative analyses require standardized methods to control and improve the analytical process in the laboratory. The availability of urine reference materials (RMs) may offer a feasible option to improve the accuracy of urine analysis and to control matrix effects. This paper presents the complete process of the development of matrix RMs in urine, including sample preparation, homogeneity, and stability studies, as well as uncertainty assessment. A freeze-drying process was developed, and freeze-dried human and pig urine samples were prepared and verified to have comparable homogeneity to liquid samples and higher stability than liquid human, pig, and artificial urine samples at 4℃ or room temperature and under extreme conditions. A total of 21 authentic urine samples from August 2022 were measured with freeze-dried RMs and spiked urine samples, and the reliability of the quantification of the RMs was compared. The freeze-dried human urine matrix RM appeared to be an excellent tool for daily quality control, as it showed high stability and gave the most consistent results with spiked samples.

Development of the certified reference material of chromium in freeze-dried human urine

To develop the certified reference material in freeze-dried human urine. Normal human urine was collected, mixed and filtered as a matrix, then the urine was prepared by adding standard, mixing, sub-packaging, and freeze-drying; After the homogeneity and stability were tested by inductively coupled plasma mass spectrometer, the urine were determined by two methods in seven independent laboratories and the uncertainty of the certified reference material was evaluated. The minimum sample intake of the developed certified reference material was 0.50 mL, and the homogeneity was good. The freeze-dried material was stable for 12 months at ≤4 ℃, and the reference material was stable at 4 ℃ after reconstitution for the 7 d. The standard value and uncertainty of the two levels were(16.0±1.0) μg/L and(30.7±2.1) μg/L(k=2), respectively. The certified reference material of chromium in freeze-dried human urine in this research met the national certified reference material requirements and could be used for the quality control of the detection of chromium in laboratory human urine.

Development of a titanium dioxide-assisted preconcentration/on-site vapor-generation chip hyphenated with inductively coupled plasma-mass spectrometry for online determination of mercuric ions in urine samples

In this study, a novel automatic analytical methodology using a titanium dioxide (TiO2)-assisted preconcentration/on-site vapor-generation (VG) chip hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) for online determination of mercuric ions (Hg2+) was developed. Interestingly, the TiO2 nanoparticle (nano-TiO2) coating on the channel surface acted not only as a sorbent for preconcentration but also as a catalyst for photocatalyst-assisted VG. Under optimum operation conditions, the developed method was validated by analyzing the certified reference material (CRM) Seronorm™ Trace Elements Urine L-2 (freeze-dried human urine). Based on the obtained results, the dramatic reduction of “hands-on” manipulation and the elimination of hazardous materials (e.g., sodium borohydride (NaBH4) and stannous chloride (SnCl2)) from the process enabled a simple and ultraclean procedure with an extremely low detection limit of 0.75 ng L−1 for Hg2+ in urine samples. To the best of our knowledge, this is the first study to report the direct exploitation of a nano-TiO2-coated microfluidic device for online sample preconcentration and on-site VG prior to ICP-MS measurement.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g−1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.

Control charts in multi-element analysis of human urine

A plan for internal quality control (IQC) was designed to monitor the quality of analytical results obtained from the simultaneous determination by ICP-MS of nine trace elements (As, Cd, Mo, Ni, Pb, U, V, W and Zr) in 1350 samples of human urine collected as part of an epidemiological study. Analytical performances, assessed during the method validation, were as follows: limit of quantification ranging from 0.002 μg/L (U) to 0.9 μg/L (Ni); recovery rates varying between 82 % (As) and 110 % (W); intermediate precision as relative standard deviation ranging from 2.5 % (Mo) to 14.2 % (V). Only for Zr, a significantly higher relative standard deviation was obtained. Initially, two commercially certified reference materials (CRMs) based on freeze-dried human urine and containing the elements of interest at suitable levels of concentration were used as control materials. However, due to considerations of reliability, convenience and cost, a control material based on fresh human urine was prepared in-house, evaluated by comparison with the CRMs and implemented for the IQC. The IQC strategy was as follows: at least one control material was analysed in each analytical session and one more control material was analysed after every ten urine samples. The central lines of the control charts were based on the assigned concentration values; the warning and action limits were set on a statistical basis, as two-times and three-times the value of the uncertainty of certified values or the observed standard deviation. At the end of the study, the data collected for the in-house control material were re-evaluated and confirmed its suitability for the purpose. The use of an in-house control material had important advantages: it was more representative of the test samples; its preparation did not involve additional steps, therefore, reducing the risk of contamination; the cost of implementing the IQC was also reduced. Some issues concerning the control charts for As and Mo were addressed and will be discussed.

Certification of steroid carbon isotope ratios in a freeze‐dried human urine reference material

An accurate method for the measurement of carbon isotope ratios of steroids in human urine has been developed at the National Measurement Institute, Australia (NMIA) for the certification of a freeze-dried human urine reference material (CRM NMIA MX005). The method measures δ(13)C values by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis following hydrolysis, solvent extraction and high performance liquid chromatography (HPLC) purification. Reference δ(13)C values for testosterone metabolites etiocholanolone, androsterone, and endogenous reference compounds (ERCs) 11β-hydroxyandrosterone and pregnanediol were determined, as well as information δ(13)C values for testosterone, epitestosterone, 11-oxoetiocholanolone, and a range of differences (Δ(13)C) between testosterone metabolites and ERCs. The measurement uncertainty was rigorously evaluated with expanded uncertainties for the reference δ(13)C values between 1.1 and 1.6 ‰ at the 95% coverage level.

Standard substance for arsenic analysis in freeze-dried human urine

目的 研制冻干人尿砷成分分析标准物质.方法 收集正常人尿液,通过过滤、混匀、分装、冷冻干燥、辐射灭菌制备而成,并对其均匀性和稳定性进行检验,通过3家实验室检测对砷标准物的不确定度进行分析.结果该标准物质均匀性良好,可稳定2年,量值及不确定度分别为(39.3±3.4)和(52.3±3.2)μg/L.结论 尿中砷成分分析标准物质各项指标符合 的要求。

An international intercomparison for 19-norandrosterone in human urine: the Comité Consultatif pour la Quantité de Matière (CCQM) Pilot Study CCQM-P68

Under the auspices of the Comite Consultatif pour la Quantite de Matiere (CCQM) a laboratory intercomparison, CCQM-P68, was co-ordinated by the National Measurement Institute of Australia (NMIA) in 2005 involving the analysis of the steroid 19-norandrosterone (19-NA) in human urine. 19-NA is the major metabolite of nandrolone, a commonly abused anabolic agent. It is routinely tested by many sports drug testing laboratories and several national metrology institutes have developed reference methods to provide reference values for the certification of reference materials. The material distributed for the intercomparison was a freeze-dried human urine candidate reference material containing 19-NA at a level close to the allowed World Anti-Doping Agency (WADA) cut-off for this anabolic agent, i.e. 2 ng/ml. Four national/designated metrology institutes participated in the intercomparison and their results for this complex analysis were in excellent agreement, with a relative standard deviation (RSD) of only 1.7%. The expanded uncertainties at the 95% level of confidence for the reported results ranged from 3.7 to 7.0% at the 2.15 ng/g level.

A high-accuracy method of analysis of 19-norandrosterone in human urine as utilised for the international laboratory intercomparison CCQM-P68

A high-accuracy exact-matching isotope dilution mass spectrometry (IDMS) method for 19-norandrosterone (19-NA) in human urine was developed at NMIA for the certification of a freeze-dried human urine reference material (CRM NMIA MX002). The method utilised GC/HRMS analysis following hydrolysis, solvent extraction, HPLC fractionation and derivatisation. The method development included investigation of all potential analytical biases for the different stages of the method and included development of a confirmatory LC/MS/MS method. The measurement uncertainty of the reference method was rigorously investigated with the achieved expanded uncertainty being less than 4% at the 95% level of confidence. The method was used in the Comite Consultatif pour la Quantite de Matiere (CCQM) pilot study CCQM-P68 which was an interlaboratory intercomparison for the analysis of 19-NA in human urine in which four national metrology institutes (NMIs) participated in. The agreement in the results from the NMIA method with those from the other NMIs was excellent.

Arsenic speciation based on ion exchange high-performance liquid chromatography hyphenated with hydride generation atomic fluorescence and on-line UV photo oxidation

An on-line method capable of the separation of arsenic species was developed for the speciation of arsenite As(III), arsenate As(V), monomethylarsenic (MMA) and dimethylarsenic acid (DMA) in biological samples. The method is based on the combination of high-performance liquid chromatograph (HPLC) for separation, UV photo oxidation for sample digestion and hydride generation atomic fluorescence spectrometry (HGAFS) for sensitive detection. The best separation results were obtained with an anion-exchange AS11 column protected by an AG11 guard column, and gradient elution with NaH2PO4 and water as mobile phase. The on-line UV photo oxidation with 1.5% K2S2O8 in 0.2 mol L(-1) NaOH in an 8 m PTFE coil for 40 s ensures the digestion of organoarsenic compounds. Detection limits for the four species were in the range of 0.11-0.15 ng (20 microL injected). Procedures were validated by analysis of the certified reference materials GBW09103 freeze-dried human urine and the results were in good agreement with the certified values of total arsenic concentration. The method has been successfully applied to speciation studies of blood arsenic species with no need of sample pretreatment. Speciation of arsenic in blood samples collected from two patients after the ingestion of realgar-containing drug reveals slight increase of arsenite and DMA, resulting from the digestion of realgar.

Application of a nitrogen microwave-induced plasma mass spectrometer as an element-specific detector for arsenic speciation analysis

A high power nitrogen microwave-induced plasma (1.3 kW) mass spectrometer (N2-MIP-MS) was successfully coupled with an HPLC system using a silica-based cation-exchange column. It was examined as an element-specific detector for its applicability to the optimization and determination of seven arsenic compounds [As(V), methylarsonic acid (MA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (TMAO) and tetramethylarsonium ion (TMI)]. The system is a promising alternative ion source for mass spectrometry for elemental speciation analysis. The MIP was stable with a pyridine mobile phase for up to 6 h. Replacing the MIP-MS fabricated nebulizer (concentric) and sample input tubing (PTFE) with an ICP-MS (PMS-2000) nebulizer (concentric) and PEEK tubing increased the ion signals for anionic and cationic arsenic compounds by 17–30 and 21–25%, respectively. PEEK tubing additionally increased the separation efficiency for the arsenic compounds. The detection limits of As(V), MA, DMA, AB, TMAO, AC and TMI obtained with the optimized HPLC-N2-MIP-MS system were 0.68, 0.95, 2.01, 0.92, 22.1, 1.31 and 1.75 µg l–1, respectively. The repeatability (RSD for three successive analyses) and reproducibility lRSD for three successive analyses performed on three different days) achieved were 0.7–9.22 and 6.5–11.4%, respectively, for the seven different arsenic compounds. No detectable spectroscopic interference of 40Ar35Cl+ was observed with a high chloride matrix (10 000 mg l–1). The developed HPLC-N2-MIP-MS method was successfully applied to the determination of arsenic compounds, principally AB, in NIES Candidate CRM-18 Freeze-Dried Human Urine (134 ± 6 µg l–1). The results agreed reasonably well with the HPLC-ICP-MS values.

Determination of Minor and Trace Elements in Urine Reference Sample by a Combined System of Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry

Simultaneous multielement determination of minor and trace elements in the urine reference sample by an ICP-MS/AES combined system is described. The biological reference sample of NIST SRM 2670 Freeze-dried Human Urine (elevated) was used for analysis. The sample preparation was kept to a minimum: the urine sample was diluted 25-fold with 0.1 M nitric acid, in which Ge, In and Tl were added as the internal standard elements. Eighteen elements at the μg/ ml-ng/ml levels were simultaneously determined by the present combined system. In addition, the present combined system was also applied to the correction of polyatomic interference in the determination of chromium in SRM 2670, where interference with 52Cr due to 40Ar12C was corrected by estimating the correction factor of 40Ar12C from correlation between the ion count of 40Ar12C and the emission intensity of carbon in the sample.

Trace elements in a commercial freeze-dried human urine reference material.

A large batch of freeze-dried human urine reference material, Seronorm Trace Elements Urine, Lot 101021, was prepared commercially (Nycomed Pharma AS, Oslo, Norway) for quality control purposes in trace element analysis. Analytes are being determined by a voluntary, international co-operative effort so that the material will be available to the scientific community at modest cost. The material is in stoppered glass vials and is to be reconstituted with 5.00 ml of water prior to use. We have evaluated the trace element content for several elements, including chromium and zinc, elements for which we have two independent methods available for the determinations, namely isotope dilution mass spectrometry (IDMS) and atomic absorption spectrometry (AAS). We also report on other trace elements measured by IDMS alone, such as Se, for which we have enriched stable isotopes available. Results for chromium indicate a mean +/- standard deviation (n = 10) of 1.2 +/- 0.3 (by IDMS) and 1.4 +/- 0.3 (by AAS) ng Cr per ml of reconstituted urine, indicating possible inhomogeneity and/or contamination (21-25% relative standard deviation, RSD). Approximately half of the observed chromium originates from the sample container. The values observed for zinc were 590 +/- 90 ng ml-1 (15% RSD) for freshly reconstituted material, 760 +/- 60 ng ml-1 (8% RSD) for material reconstituted 4 d earlier, and 940 +/- 60 ng ml-1 (6% RSD) 2 months after reconstitution. Selenium values by IDMS were very reproducible, with a mean concentration of 16 +/- 0.15 ng g-1 (0.9% RSD), suggesting little or no contamination and a high degree of sample homogeneity for this element. The source of potential contaminants has been evaluated by multielement determinations of leachates of the vials and stoppers. Elements noted in significant amounts include B, Ba, Sr, Mo, Cu and Zn, with most of the zinc coming from the rubber stopper.

Development and Characterization of an Elisa for Cotinine in Biological Fluids

AbstractCotinine is a major metabolite of nicotine and serves as an important biomarker of tobacco smoke exposure. To monitor exposure to tobacco smoke or nicotine, a sensitive enzyme-linked immunosorbent assay (ELISA) for cotinine was developed. The test had an 1–50 of between 0.5 and 1.0 ng/ml for cotinine and about 500-fold less affinity for nicotine. Few matrix effects were not detectable in human saliva, although relatively small matrix effects (1–50 for cotinine, 2.8 ng/ml) were observed in human serum and urine. The test accurately measured the levels of cotinine in NI5T standards in freeze-dried human urine derivative material (r = .9999) indicating its reliability for measurement of cotinine. The test readily detected low levels (5–500 nglml) of cotinine in human saliva and serum samples. Also, the levels of cotinine in plasma and urine samples from smoke-exposed mice and rats could be rapidly analyzed for cotinine. This ELISA is therefore a sensitive and accurate test for the determination of co...

The certification of cocaine and benzoylecgonine in a human urine standard reference material.

The concentrations of cocaine and benzoylecgonine (BE) in Standard Reference Material (SRM) 1508, cocaine and metabolites in freeze-dried human urine, were determined at the National Institute of Standards and Technology (NIST, formerly NBS) by two independent methods. For cocaine, one method was based on gas chromatography/mass spectrometry (GC/MS); the other was based on high-performance liquid chromatography (HPLC). For BE, one method was based on GC/MS; the other was based on flow injection analysis/thermospray mass spectrometry (FIA/MS). The results for each pair of methods were statistically evaluated. Concentrations were determined in the SRM for three levels of cocaine and three levels of benzoylecgonine. Methylecgonine, although present in the material, was not determined. For cocaine, the concentrations were 90, 263, and 429 ng/mL of human urine. For BE, the concentrations were 103, 259, and 510 ng/mL of human urine.