Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L⁻¹. Over the concentration range investigated (0.05–10 mg L⁻¹), the calibration curve was obtained with satisfactory linearity (R² = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.     

Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.     

Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe⁺), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe⁺ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled ¹³CD₃⁸²SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe⁺, the standard addition method was applied. Quality control for the accurate quantitation of TMSe⁺ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L⁻¹. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L⁻¹ for TMSe⁺, 5.6 μg Se L⁻¹ for SeMet, and 0.1 μg Se L⁻¹ for both SeGalNAc and SeGluNAc.     

A solid-phase extraction and size-exclusion liquid chromatographic method for polyethylene glycol 25 p-aminobenzoic acid determination in urine: validation for urinary excretion studies of users of sunscreens

No previous publications about percutaneous absorption of polyethylene glycol 25 p-aminobenzoic acid (PEG-25 PABA) have been found in the literature and the expected levels to be found in human urine after sunscreens use are unknown. The method proposed here is suitable to determine PEG-25 PABA in the urine of sunscreens users in order to carry out studies on body accumulation/excretion. It is based on solid-phase extraction (SPE) with size-exclusion liquid chromatography determination. Solid-phase extraction allows the analyte to be retained and subsequently eluted for a clean-up, using a silica-based cartridge. The size-exclusion liquid chromatography of the eluted allows the rest of matrix interferences to be avoided. Fluorescence intensity was measured at λ_em = 350 nm (λ_exc = 300 nm). The sensitivity of the proposed method is in the order of 450 ± 5 mL ng⁻¹ and the detection limit (3 S_y/x/b) in the measured solutions is in the order of 13 ng mL⁻¹, that is 2.6 ng mL⁻¹ in urine samples. The method enables PEG-25 PABA to be determined in both, spiked and unspiked human urine samples. Results obtained for spiked human urine samples (11-100 ng mL⁻¹) demonstrated the accuracy of the method. The mean relative standard deviation of the results was in the order of 3-10%. Three volunteers applied a sunscreen lotion containing a 8% PEG-25 PABA sunscreen cream and their urinary excretion was controlled from the moment of application until the excreted amounts were no longer detectable.     

Liquid chromatography tandem mass spectrometry determination of free and conjugated estrogens in breast cancer patients before and after exemestane treatment

We report liquid chromatographic separation with tandem mass spectrometry determination of 12 endogenous estrogens and their intact conjugates in blood and urine and its application to study effects of exemestane treatment on estrogen generation and metabolism in postmenopausal women with estrogen-dependent breast cancer. A 0.5 mL aliquot of each urine or serum sample is fractionated with solid phase extraction to a fraction of free estrogen and another fraction of their conjugates. The reversed phase LC/MS/MS determines dansylated estrogens with positive ionization and intact conjugates with negative ionization. The method provides reproducible separation and limit of detection as low as 1 pg mL⁻¹ for free estrogens and 0.03 ng mg⁻¹ creatinine for the conjugates in serum and urine samples. The method enabled us to acquire unique concentration profiles of 12 endogenous estrogens and their intact conjugates in 30 breast cancer patients before and after one month of exemestane treatment. Exemestane suppressed total serum and urinary estrogens by 11–97% (P < 0.0001) and 8.7–91% (P < 0.0001), respectively. Specifically, these data show that exemestane preferentially suppressed E1, E1-3S, E1-3G, and E2-17G more than other estrogens. Linear regression analysis of estrogen concentrations before and after treatment showed correlation coefficients of 0.8385 (n = 289, P < 0.0001) and 0.8863 (n = 360, P < 0.0001). This study provides urinary and blood estrogen concentration profiles in breast cancer patients to demonstrate the effect of exemestane on estrogen generation, supporting inhibition of aromatase activity.     

Improved methods for urinary atrazine mercapturate analysis--assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis^® HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL⁻¹), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis^® HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL⁻¹) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R² values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R² = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.     

Method optimization for quantitative analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by liquid chromatography-electrospray ionization mass spectrometry

A simple, sensitive LC-ESI-MS method was optimized for quantitative analysis of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in environmental samples. Under negative ionization mode, HMX can form adduct ions with various organic acids and salts, including acetic acid, formic acid, propionic acid, ammonium nitrate, ammonium chloride, sodium nitrite, and sodium nitrate. Acetic acid was chosen as additive and the ion, [M + CH₃COO]⁻ with m/z = 355 was used for selective ion monitoring (SIM) in this study. Good sensitivity was achieved with low acetic acid concentration in the mobile phase and relatively low capillary temperature. The method detection limit was 0.78 pg for HMX in standard solution. Linearity (R² > 0.9998) was obtained at low concentrations (0.5-50 μg/L). This method has been used to determine HMX concentrations in water samples and lizard egg samples from an animal exposure study.     

Rapid Folin-Ciocalteu method using microtiter 96-well plate cartridges for solid phase extraction to assess urinary total phenolic compounds, as a biomarker of total polyphenols intake

Nutritional markers have several advantages for epidemiologic and clinical assays, when compared to dietary data obtained by food frequency questionnaires. Few studies have assessed whether total polyphenol (TP) compounds provide a valid biomarker for TP intake. To date, there has been almost no literature describing methods to determine TP in complex matrices such as urine, which have many interfering substances.We report a rapid Folin-Ciocalteu method to determine TP in urine samples using Oasis^® MAX 96-well plate cartridges for solid phase extraction. These plates allow analysis of a high number of samples at the same time. We performed a prospective, randomized, crossover trial and one cross-sectional study with 60 volunteers from the PREDIMED trial, seeking to evaluate whether the TP in urine were correlated with polyphenol intake and could, therefore, be considered as a marker of intake of these compounds.The assay was optimized; the sensitivity and the polarity range of urine polyphenols were increased and the detection and quantification limits were significantly reduced. The metabolites in standards solution and urine samples were stable under the storage and handling conditions. In the clinical trial and the cross-sectional study, TP excreted in spot urine samples were positively correlated with TP intake, r = 0.48, P < 0.01 and r = 0.257, P = 0.04, respectively.The methodology described may be used to detect TP in urine samples, employing the high throughput of 96-well microtiter plates and reader. The method is fast and simple and it allows analysis of a large number of samples at the same time.     

Use of ion mobility spectroscopy with an ultraviolet ionization source as a vanguard screening system for the detection and determination of acetone in urine as a biomarker for cow and human diseases

An ion mobility spectrometer equipped with an ultraviolet lamp was used for the qualitative and quantitative determination of acetone in urine samples. This analyte can be used as a biomarker for some fat metabolism-related diseases in humans and cows. Samples require no pretreatment other than warming at 80 °C for 5 min, after which an N₂ stream is used to drive volatile analytes to the ion mobility spectrometer. The precision of the ensuing method, expressed as relative standard deviation (%RSD), is better in all cases than 6.7% for peak height and calculated at three levels of concentration. The analyte concentration range studied was from 5 to 80 mg L⁻¹, its limit of detection in the aqueous matrix 3 mg L⁻¹ and recoveries from spiked urine samples 109 ± 3%. The calculated reduced mobility for acetone in the urine samples, 1.75 ± 0.04 cm² V⁻¹ s⁻¹, was similar to previously reported values. Also, the results were consistent with those provided by test strips used for reference. The proposed method provides a new vanguard screening system for determining acetone in urine samples.     

Simultaneous determination of urinary hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid for biomonitoring of volatile organic compounds by gas chromatography-mass spectrometry

A sensitive and precise method for the simultaneous determination of hippuric acid, o-, m- and p-methylhippuric acids, mandelic acid and phenylglyoxylic acid, which are major urinary metabolites of toluene, o-, m- and p-xylenes, styrene and ethylbenzene, respectively, was developed. These metabolites were converted into their methyl ester derivatives with methanol in hydrochloric acid, and then quantitated by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring using a DB-1 capillary column. The injected compounds were quantitatively and reproducibly resolved within 19 min with a detection limit of 8-27 pg. The calibration curves were linear in the range of 0.05-25 μg for each compound, with correlation coefficients above 0.9999. This method was successfully used to analyze small amounts of both rat and human urine samples without any interference from coexisting substances. Overall recoveries of these compounds spiked in urine samples were 92-104%. The analytical results of the contents of these metabolites in the rat and human urine samples are presented.     

Portable microfluidic system for determination of urinary creatinine

A simple, low cost and portable microfluidic system based on a two-point alkaline picrate kinetic reaction has been developed for the determination of urinary creatinine. The creatinine reacts with picric acid under alkaline conditions, forming an orange-red colour, which is monitored on PDMS microchip using a portable miniature fibre optic spectrometer at 510 nm. A linear range was displayed from 0 to 40 mg L⁻¹ creatinine (r² = 0.997) with a detection limit of 3.3 mg L⁻¹ (S/N = 3). On-chip absorbance signals are reproducible, with relative standard deviations (RSDs) of 7.1%, when evaluated with 20 mg L⁻¹ creatinine (n = 10). The standard curves in which the intra-run CVs (4.7-6.8%) and inter-run CVs (7.9%) obtained were performed on three different days and exhibited good reproducibility. The method was highly correlated with the conventional spectrophotometric method when real urine samples were evaluated (r² = 0.948; n = 15).     

Enantiomeric quantification of (S)-(+)-methamphetamine in urine by an immunoaffinity column and liquid chromatography-electrospray-mass spectrometry

A method using an immunoaffinity column (IAC) and liquid chromatography-electrospray ionization mass spectrometry (LC/MS) for on-line detecting the presence of MA in the effluent was developed for the quantitative and enantiomeric determination of (S)-(+)-methamphetamine (d-MA) in urine. The IAC was made in our laboratory and utilized in the LC/MS to simultaneously extract and separate enantiomers of MA from urine samples. An aqueous ammonium acetate buffer was used as the mobile phase. Urine samples were spiked with racemic deuterated methamphetamine (MA-d₁₄) as internal standard (IS), filtered through a membrane, and injected into the LC/MS without any further pre-treatment. Protonated molecular ion of MA and MA-d₁₄ (m/z 150 and 164) were isolated and further fragmented, the respective product ions, m/z 119 and 130, were collected for quantitative determination. This is an improvement of our previous method (A.C. Lua, Tsong-Yung Chou, J. Chromatogr. A 967 (2002) 191). In the previous method, MA was separated with HPLC, the efflux was fractionated and each fraction was either determined with an immunoassay or GC/MS. Monitoring of MA in the efflux is tedious and time consuming. Urine samples spiked with different concentrations of d-MA were measured by this method. A linear relationship exists in the 150-1050 ng/mL range, and the detection limit (defined as signal-to-noise ratio 3) of d-MA was determined to be 18 ng/mL. The linearity of the method for d-MA can be described by the equation (Y = 1.415 × 10⁻³X + 0.034, correlation coefficient: r² = 0.999). Within run, accuracy and precision (n = 6, relative error: −7.2 to +4.0% and relative standard deviation: 3.8-9.3%) of the method are fairly good.     

Fluorimetric determination of phytic acid in urine based on replacement reaction

A sensitive fluorimetric method for determination of phytic acid in human urine samples was described. The method was based on a fluorimetric replacement reaction, in which the added phytic acid replaced the Cu²⁺ ion from Cu²⁺-gelatin complex, liberating the fluorescent gelatin molecule. The fluorescence of the solution was accordingly recovered proportionally to the amount of the foreign phytic acid. The excitation wavelength was 273.5 nm and the characteristic emission wavelength was 305.0 nm, respectively. The calibration graph was obtained by plotting the recovered fluorescent intensity at maximum 305.0 nm against the added standard phytic acid, and was divided into two sections. One section was linear over the range of 0.40-2.40 mg L⁻¹ with a linear regression equation of I_f = −0.895 + 15.146c (R² > 0.9993), and the other over the range of 2.40-9.20 mg L⁻¹ with a linear regression equation of I_f = −29.526 + 26.113c (R² > 0.9996), respectively. The relative standard deviation (R.S.D.) at 95% confidence degree for a 2.0 mg L⁻¹ of standard phytic acid within 1 month was less than 1.26% (n = 5), indicating the procedure is reproducible. The detection and the quantification limits of phytic acid were estimated to be 0.23 and 0.40 mg L⁻¹, respectively. The proposed method was applied to the determination of phytic acid in urine samples and the found concentrations of phytic acid in urine were in the range of 0.49-0.75 mg L⁻¹ with recoveries of 96.2-108.8%. Comparison of the obtained results with the reported HPLC was performed, indicating the proposed method was reliable.     

The potential of autofluorescence spectroscopy to detect human urinary tract infection

Urinary tract infections (UTIs) are known to alter the normal urine composition which, in principle, can lead to changes in urine autofluorescence. This paper describes the study of human urine (normal and UTI) by using UV fluorescence excitation/emission matrices and synchronous spectra and proposes a method of diagnosing UTI without any sample preparation. The method is based on excitation in the shorter UV region (250-350 nm) which shows good discrimination between the normal urine and UTI samples. The synchronous scans with an offset of Δλ = 90 nm were also able to differentiate between normal urines and UTI samples. These differences were observed even though the two known major urine fluorophores, tryptophan and indoxyl sulfate were present in the normal urine and UTI samples in similar concentration as established by HPLC analysis. Although the identity of substances responsible for the altered autofluorescence in UTI is not established, our study shows that autofluorescence has the potential to differentiate between normal human urine samples and those with UTI.     

Simple sequential injection analysis system for rapid determination of microalbuminuria

A simple, specific and sensitive sequential injection analysis (SIA) system based on non-immunoassay fluorescent detection has been developed for the determination of urinary albumin. The specific binding of the dye Albumin Blue 580 (AB 580) to albumin in urine generated high emission fluorescent signals. The excitation and emission wavelengths were set at 590 and 610 nm, respectively. The analytical range was obtained from 1 to 100 mg L⁻¹, with a detection limit of 0.3 mg L⁻¹ (S/N = 3). The SIA system gave high precision with relative standard deviations (R.S.D.s) of 0.9% and 1.4% when evaluated with 15 and 100 mg L⁻¹ albumin (n = 15), respectively. The method exhibited good reproducibility, as assessed by performing four analytical curves on different days, and intra-run CVs (2.3-3.3%) and inter-run CVs (3.8%) were obtained. Rapid operation was achieved with a sample throughput of 37 h⁻¹. This method was successfully applied to the determination of urinary albumin, and the method was highly correlated with the immunoturbidimetric method (r² = 0.965; n = 72).     

Validation of an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and S-phenylmercapturic acid

The aim of this study is to validate isotope-dilution electrospray ionization tandem mass spectrometry (ESI-MS-MS) method with a dual-loop cleanup device for simultaneous quantitation of two benzene metabolites, trans, trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA), in human urine. In this study, a pooled blank urine matrix from rural residents was adopted for validation of the analytical method. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of ttMA and SPMA standards spiked into two kinds of urine matrixes over a wide concentration range, 1/32-8-fold biological exposure indices (BEIs) values, showed good linearity (R > 0.9992). The detection limits in pooled urine matrix for ttMA and SPMA were 1.27 and 0.042 μg g⁻¹ creatinine, respectively. For both of ttMA and SPMA, the intra- and inter-day precision values were considered acceptable well below 25% at the various spiked concentrations. The intra- and inter-day apparent recovery values were also considered acceptable (apparent recovery >90%). The ttMA accuracy was estimated by urinary standard reference material (SRM). The accuracy reported in terms of relative error (RE) was 5.0 ± 2.0% (n = 3). The stability of sample storage at 4 or −20 °C were assessed. Urinary ttMA and SPMA were found to be stable for at least 8 weeks when stored at 4 or −20 °C. In addition, urine samples from different benzene exposure groups were collected and measured in this system. Without tedious manual sample preparation procedure, the analytical system was able to quantify simultaneously ttMA and SPMA in less than 20 min.     

Simple and sensitive liquid chromatographic method with fluorimetric detection for the analysis of gamma-amino-n-butyric acid in human urine

A simple and sensitive liquid chromatographic method is described for the analysis of γ-amino-n-butyric acid (GABA) in human urine. GABA is increased in the urine of cancer patients and could be used as a biomarker in the diagnosis and treatment of related patients. The method is based on derivatizing GABA with a fluorescent reagent (naproxen acyl chloride) for transforming the non-chromophoric GABA to a derivative with chromophoric and fluorophoric properties. The resulting derivative is highly responsive to a fluorimetric detector (λ_ex = 230 nm, λ_em = 350 nm). The lower quantitation of the method is attainable at 100 nM GABA with a detection limit about 10 nM (S/N = 3 with 20 μL injected). Application of the method to the analysis of GABA in the urine of patients with ovarian and uterine cancer was demonstrated.     

Voltammetric determination of Imatinib (Gleevec) and its main metabolite using square-wave and adsorptive stripping square-wave techniques in urine samples

The voltammetric behaviour of Imatinib (STI 571) and its main metabolite (N-demethylated piperazine derivative) were studied by square-wave techniques, resulting in to two methods for their determination in aqueous and urine samples at pH 2. The application of the square-wave (SW) without the adsorptive accumulation and voltammetric stripping (AdSV) exhibit a peak at a reduction potential of −0.70 V for an accumulation potential of −0.45 V. The sensitivity was higher for the stripping technique because a signal four times higher than that provided by the square-wave method without the previous accumulation was obtained. Due to the fact that Imatinib and its metabolite show the same voltammetric reduction process, some experiments were performed in order to compare the voltammetric response of Imatinib and its main metabolite in a similar ratio than that of the therapeutic concentration. The calibration curve for Imatinib in urine was linear in the range from 1.9 × 10⁻⁸ to 1.9 × 10⁻⁶ M in stripping mode with an accumulation time (t_acc) of 10 s. The relative standard deviations obtained for concentration levels of Imatinib as low as 2.0 × 10⁻⁷ M for square-wave was 2.17% (n = 9) and for stripping square-wave was 2.65% (n = 9) in the same day. The limits of detection for square-wave and stripping square-wave were 5.55 × 10⁻⁹ and 5.19 × 10⁻⁹ M, respectively. Thus, the presented method are straightforward, rapid and sensitive and has been applied to the determination of Imatinib and its main metabolite altogether in urine samples from real patients.     

Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum

We present a method for the simultaneous determination of guanidinosuccinic acid (GSA) and guanidinoacetic acid (GAA) from urine by protein precipitation and liquid chromatography/tandem mass spectrometry. The chromatographic separation was performed using a cation exchange column with an elution gradient of 0.1 mM and 20 mM ammonium acetate buffers. GSA was detected with the mass spectrometer in negative ion mode monitoring at m/z 174.1, and GAA, creatinine, arginine, and homoarginine were in positive ion mode monitoring at m/z 118.1, 114.1, 175.1, and 189.1, respectively. As an internal standard, l-arginine-¹³C₆ hydrochloride and creatinine-d₃ (methyl-d₃) were used. The calibration ranges were 0.50-25.0 μg mL⁻¹, and good linearities were obtained for all compounds (r > 0.999). The intra- and inter-assay accuracies (expressed as recoveries) and precisions at three concentration levels (1.00, 5.00 and 25.0 μg mL⁻¹) were better than 83.8% and 7.41%, respectively. The analytical performance of the method was evaluated by determination of the compounds in urine from male C57BL/J Iar db/db diabetes mellitus (DM) mice. The values of GSA and GAA corrected by the ratios of the individual compounds to creatinine were significantly increased in DM mice compared with control mice. These results indicated that the newly developed method was useful for determining urinary guanidino compounds and metabolites of arginine.