Fully Enzymatic Colorimetric Assay of Serum and Urine Creatinine Which Obviates the Need for Sample Blank Measurements

Abstract

A fully enzymatic method for the colorimetric determination of serum and urine creatinine is described which does not require sample blank measurements. It is based on the formation of hydrogen peroxide from creatinine in a reaction sequence catalyzed by creatinine iminohydrolase, ATP-dependent 1-methylhydantoinase, N-carbamoylsarcosine amidohydrolase and sarcosine oxidase. The hydrogen peroxide is quantitated with high sensitivity at 546 nm by a chromogenic system consisting of peroxidase, 2′-sulpho-2-methyl-benzthiazolinone hydrazone and 2,4,6-tribromo-3-hydroxy-benzoic acid. Only 20 μL of sample are needed for the assay, the total reaction being complete within 10 min at 25°. Within-run precision gave a CV of 3.1 and 1.6 % at serum creatinine concentrations of 79 and 160 μmol/L, respectively, and the standard curve is linear up to at least 1760 μmol/L. The assay yields results which agree well with those found by both an enzymatic UV-method and an alternate enzymatic colorimetric procedure necesitating sample blank measurements to correct for endogenous creatine.     

A 31P NMR Study of the Stability of Carboxyifosfamide and Carboxycyclophosphamide,Two Metabolites of Ifosfamide and Cyclophosphamide

Abstract

Ifosfamide (IF) and cyclophosphamide (CP) are two phosphorated anticancer agents used in the treatment of solid tumours. Several phosphorated metabolites, among them carboxyifosfamide (CXIF) and carboxycyclophosphamide (CXCP), were detected and quantified by ³¹P NMR in urine from patients treated with IF or CP. In agreement with other authors [1], we observed a great inter-patient variability in the urinary excretion of CXIF in patients treated with IF [2]. This variability was attributed to a genetic polymorphism of aldehyde dehydrogenase, the enzyme responsible for the formation of CXCP or CXIF [1,3]. Since CXCP and CXIF are unstable, we thought that the inter-individual variability could also be due to a degradation during the storage of urine samples. A ³¹P NMR study of the stability of CXIF and CXCP in urine as a function of time, pH (7 and 5.5) and storage temperature (25°C, 8°C, ?20°C, ?80°C) demonstrated that (i) CXCP and CXIF are more stable at pH 7 than at pH 5.5, (ii) CXCP is more stable than CXIF at both pH, (iii) the degradation decreases with temperature but still occurs at ?20°C and even ?80°C. For an accurate quantification of these compounds, the storage of urine samples must be done at ?80°C immediately after collection and not exceed 1 month at pH 7 whereas, at pH 5.5, the assay must be carried out in the few days following the sampling. To identify the degradation products of CXCP and CXIF, the time course of hydrolysis (between pH 2 and 7) of these compounds was monitored by ³¹P NMR. The structure of each compound formed was determined by mass spectrometry and ¹H and ¹³C NMR after their isolation (except compound A too unstable to be isolated). The results are reported in the following schemes.     

Rapid High Performance Liquid Chromatographic Determination of Ampicillin in Human Urine

Abstract

A rapid, sensitive and specific HPLC assay for the determination of ampicillin in human urine is developed.

Ampicillin was directly measured in human urine at 225 nm using a reversed phase column (Synchropack RP-P) and a mobile phase composed of (1:9 methanol-sodium acetate solution, 0.01 M, pH 4). The analysis required no longer than 10 min. Linear correlation between the peak height ratio of ampicillin to cefoxitin sodium (internal standard) and ampicillin concentration in urine over the range 10–100 μg ml^?1 was obtained. The developed method proved to be advantageous as it monitors ampicillin level in urine. Moreover, the urinary excretion of ampicillin in human subjects after an oral administration of 500 mg ampicillin capsules was established using the proposed method.     

The Determination of Ioglycamic Acid in Bile and in Urine by High Performance Liquid Chromatography

Abstract

A fast, simple and specific method for the determination of ioglycamic acid in bile and in urine is described. Reversed-phase ion-pair chromatography with methanol-water (67.5: 32.5) in the presence of the counter ion tetrabutyl-ammonium phosphate as the mobile phase separates ioglycamic acid in 5 min at a flow rate of 1 ml/min. Under the same conditions urinary creatinine can be simultaneously determined and may be used as an endogenous internal standard. Bile and urine from a healthy subject were screened for possible metabolites but these are not detected.     

Determination of the New Ace-Inhibitor Quinapril and Its Active Metabolite Quinaprilate in Plasma and Urine by High-Performance Liquid Chromatography and Pre-Column Labelling for Fluorescent-Detection

Abstract

A sensitive and selective method for the determination of quinapril and its active metabolite quinaprilate in human plasma and urine is described. The method is based on isolation using C18 Bond Elut cartridges, pre-column derivatization with 9-anthryldiazo-methane and purification of the reaction mixture on CBA columns followed by reversed-phase high performance liquid chromatography with fluorometric detection. Calibration curves were linear between 20 ng and 1000 ng/ml of plasma (100-2000 ng for urine) for both substances, the lower limit of detection being 5-10 ng/ml.

The present assay procedure has been applied to monotoring plasma and urine concentrations in several pharmacokinetic studies in humans.     

Determination of Urinary Bis(2-ethylhexyl)phthalate Levels in Non-uremic Subjects by Gas Chromatography

Abstract

In vivo studies of urinary bis(2-ethylhexyl)phthalate (DEHP) levels in dogs and in non-uremic patients undergoing hemodialysis treatments for psoriasis were undertaken. Dogs were divided into 3 groups: Control, Sham-Operated, and Nephrectomized. Each dog received 225 mg DEHP per kilogram body weight via the femoral vein. Each of the non-uremic patients underwent hemodialysis therapy for 4–5 hours once a week for four consecutive weeks to treat their psoriatic condition. Specimens of 2 4 hr urine were collected and analyzed for DEHP by gas chromatography. The detection limit of DEHP in urine is 15 ng/ml. No detectable DEHP was found in the urine of all pre-injection specimens obtained from all three groups of dogs. The total urinary DEHP concentrations for the four day period were found to be 76.1 and 192.2 μg for the Control and the Sham-Operated dogs, respectively. No urine samples could be collected from the Nephrectomized dogs. DEHP levels were found in the 24 hr urine specimens from some of the non-uremic patients undergoing hemodialysis therapy. The DEHP concentrations ranged from non-detectable to 159.8 yg/24 hrs. Normal renal function seems to be necessary for the excretion of non-metabolized DEHP.     

Quantitation of Creatinine in Urine and Plasma Samples by Reversed Phase HPLC

Abstract

Creatinine determination in urine and plasma affords an index of the renal function. Reversed-phase high pressure liquid chromatography was used for the separation and quantitation of creatinine in normal and arsenic exposed human urine samples. Acetonitrile/water (1:1) was the mobile phase. The method was compared with the Jaffé alkaline picrate reaction. Results show that the HPLC procedure has high reproducibility and samples are stable at the storage conditions. Plasma samples required depro-teinization and extraction with CH₃CN prior to HPLC analysis, while urine samples required only centrifugation.     

A Simple Isocratic HPLC Method for the Determination of Metoclopramide in Plasma and Urine

Abstract

Metoclopramide concentrations in plasma and urine were determined by high performance liquid chromatography using a cyanopropylsilane column and UV detection. The mobile phase consisted of 0.03M sodium acetate (pH 7.4) and acetonitrile. The plasma samples were extracted with dichloromethane after pH adjustment. Urine proteins were precipitated with acetonitrile. The reproducibility and precision of the methods were demonstrated by the analysis of samples containing 5 – 200 ng/ml plasma and 0.25 – 200 ug/ml urine.

The glucuronide and sulfate conjugates of metoclopramide were also quantitated after differential acid hydrolysis of urine samples. The conditions for acid hydrolysis were studied. The methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

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.     

Some aspects of quantification of neutral steroid metabolites in body fluids of healthy and uremic subjects by capillary gas chromatography

Neutral steroid metabolites enriched from urine and hemofiltrate were identified by gas chromatography/mass spectrometry and quantified by capillary gas chromatography. This study included 20 healthy controls and 37 uremic patients. Before enrichment of steroids from biological material, the standard deviation of the workup procedure and subsequent derivatization into the trimethylsilyl-enol-trimethylsilyl ethers was tested and found to be 2–5% in urine and 12–17% in the more complicated workup procedure of hemofiltrate, but essentially smaller than the biological standard deviation. Compared to the 24 h urinary excretion rates of controls, the excretion rates of androsterone, etiocholanolone, and corticoid metabolites were significantly lower in uremic body fluids, while those of 11-oxygenated androstanolones, degradation products of corticoids, were enhanced in uremic urine. The ratio of corticoid metabolites to 11-oxygenated androstanolones in urine of nondialyzed uremics correlated significantly with their plasma creatinine levels.     

Monitoring of Fluorescent-Labelled Main Progesterone Metabolite in Marmoset Monkeys

Abstract

5α-pregnane-3α, 7α-diol-20-one, the main progesterone derivative in marmoset monkeys, shows a very low absorption in UV-light. Labelling of this metabolite prior to HPLC and subsequent monitoring by an UV-detector is essential. We used dansyl-hydrazine for the formation of a fluorescent derivative. The optimum conditions for the reaction of dansyl-hydrazone with hydroxypregnanolone were controlled by HPTLC, chromatographic separation is carried out by HPLC and HPTLC and is described in this paper. When the quantitation of the dansylhydrazones is carried out by HPTLC, the fluorescence intensity of the derivative can be increased by a factor up to 5 by dipping the HPTLC plate into a mixture of paraffin/n-hexane or after treatment with triethanolamin/isopropanol (1:4; by vol). Hydroxypregnanolone from marmoset urine is detected for the first time by liquid chromatography. The derivative is of practical use to determine ovulation and pregnancy in the marmoset monkey. Quantitation by HPLC and thin-layer chromatography is possible in the range of 10 to 1000 ng. The concentrations in marmoset urine during the luteal phase is in the range of 50 to 400 ng/mg creatinine.     

GC-MS Derivatization Studies: The Formation of Dexamethasone MO-TMS

Abstract

The 16α-methyl group in dexamethasone increases drastically the steric hindrance to reaction of both the 20-ketone and 17α-hydroxyl groups, as shown by kinetic studies with GC-MS techniques. A procedure is described for the preparation of the MO derivative (reaction et 60[ddot]C for 3 hr) and complete conversion of all hydroxyl groups to TMS ether groups (reaction at 100[ddot]C for 6 hr). The resulting MO-TMS derivative is thermostable and suitable for use in GC-MS methods. The procedures usually employed in urinary steroid studies are satisfactory for prednisone and prednisolone.     

Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine

A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex—a cannabis plant extract containing 1:1 ?⁹-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS. Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units β-glucuronidase from Red Abalone. Calibration curves were fit by linear least squares regression with 1/x ² weighting with linear ranges (r ²?>?0.990) of 2.5–100 ng/mL for non-hydrolyzed CBD and 2.5–500 ng/mL for enzyme-hydrolyzed CBD. Bias was 88.7–105.3 %, imprecision 1.4–6.4 % CV and extraction efficiency 82.5–92.7 % (no hydrolysis) and 34.3–47.0 % (enzyme hydrolysis). Enzyme-hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration.     

The Mechanisms of Acid-Catalyzed Hydrolysis of N-(4-Substituted Arylthio) Phthalimides

Abstract

The acid-catalyzed hydrolysis of N-(4-substitutedarylthio)phthalimides was studied in aqueous solutions of sulfuric, perchloric, and hydrochloric acids at 40.0 ± 0.1 °C. Analysis of the data by the excess acidity method, activation parameters, and substituent effects indicates hydrolysis by an A-2 mechanism at low acidity. At higher acidities, a changeover to an A-1 mechanism is observed.

GRAPHICAL ABSTRACT     

Comparison of 24-h volume and creatinine-corrected total urinary polyphenol as a biomarker of total dietary polyphenols in the Invecchiare InCHIANTI study

Polyphenols have beneficial effects on several chronic diseases but assessing polyphenols intake from self-reported dietary questionnaires tends to be inaccurate and not very reliable. A promising alternative is to use urinary excretion of polyphenols as a proxy measure of intake. The best method to assess urinary excretion is to collect 24-h urine. However, since collecting 24-h urine method is expensive, time consuming and may be difficult to implement in large population-based studies, measures obtained from spot urine normalized by creatinine are commonly used. The purpose of the study was to evaluate the correlation between polyphenols dietary intake and total urinary polyphenol excretion (TPE), expressed by both 24-h volume and urinary creatinine normalization in 928 participants from the InCHIANTI study. Dietary intake data were collected using a validated food frequency questionnaire. Urinary TPE was analyzed by Folin-Ciocalteau assay. Both urinary TPE expression models were statistically correlated (r=0.580), and the partial correlation coefficient improved (pr=0.722) after adjusting for the variables that modify the urinary creatinine excretion (i.e. gender, age, BMI, physical activity and renal function). In crude models, polyphenol intake was associated with TPE corrected by 24-h volume (r=0.211; P<0.001), but not with creatinine normalization (r=0.014; P=0.692). However, urinary TPE expressed by creatinine correction was significantly correlated with dietary polyphenols after adjusting for covariates (pr=0.113; P=0.002). We conclude that urinary TPE expressed by 24-h volume is a better biomarker of polyphenol dietary intake than by urinary creatinine normalization. After covariate adjustment, both can be used for studying the relationships between polyphenol intake and health in large-scale epidemiological studies.     

Enzymatic Detection of Urinary Steroid-17β-Glucuronides after Gel Filtration

Abstract

An enzymatic detection of urinary steroid-17β-glucuronides is described. The principle of the method is as follows; after gel filtration with Sephadex G-25 β-glucuronidase is added to each effluent fraction and incubated for 20 h at 37 $C. After hydrolysis, 3β, 17β-hydroxysteroid dehydrogenase is added and incubated for 20 min at 37 $C. An absorbance at 500 nm is read aginst sample of first fraction effluent.     

Photochemical behaviour of Schiff base liquid crystals based on isoxazole and isoxazoline ring. A kinetic approach

ABSTRACT

The kinetic study on the hydrolysis of Schiff bases (SBs) 1a-c and 2a-c induced by UV-vis was undertaken as a complementary study of the stability in solution and in bulk of the SBs. Solutions in chloroform were exposed to UV-vis and acquisition data occurred for 1a-c and for 2a-c in 21°C, 30 °C, 35°C and 40°C. Kinetic profile for 1a-c and 2a-c displayed the similar photochemical behaviour in that four temperature values. At 21°C, two kinetic regimes were observed where the decomposition of SBs is faster at the initial stage, with no linear plot of absorbance vs time, and after the kinetic profile obeyed a linear behaviour. A mathematical treatment of the experimental data was applied, which allowed associating the initial stage data with a second-order reaction, and the final stage of the hydrolysis with a first-order reaction. The mechanism of photochemical hydrolysis of SBs 1a and 2a was addressed. It was composed of three parts, the excitation, then the isomerisation and activation processes of tetrahedral intermediate and, the last process, a collapse of the intermediate to the yielded products of the hydrolysis aldehydes and amines which were detected by their UV-vis spectrum.     

Excretion of biotrace elements using the multitracer technique in mice

A radioactive multitracer solution obtained from the nuclear reaction of selenium with 25 MeV/nucleon⁴⁰Ar ions was applied to the investigation of the trace elements behavior in feces and urine of mouse. The excretion rates of 23 elements, Na, K, Rb, Mg, Ca, Sr, Ga, As, Sc, V, Cr, Mn, Co, Fe, Zn, Y, Zr, Mo, Nb, Tc, Ru, Ag and In were simultaneously detected under strictly identical experimental conditions, in order to clarify the excretion behavior of the elements in mice. Fecal and urinary excretion rates of the elements in mice reached the highest value separately at 48 and 24 hours. The total excretion of Mo, Tc and Co within 96 hours were all larger, more than 60%. Accumulative excretion rates of Ca, Nb, Mg, Sr, V, Sc, Na, Cr, Fe, Ag, Mn and Zr were 60-30%. The total rates of Ru, K, As, Zn, Rb, Y, Ga and In were less than 30%, and low excretion. The main excretion pathway of Mo, Co, Mg, Fe and Ag was through urine, and Na, K, As and Rb were eliminated from the body also in urine. But fecal excretion of Tc, Nb, Sr, Y, Ru, and In were larger than urinary excretion, and Ca, Sc, Mn, Zr, Zn were eliminated from the body in feces.     

Optimization in the Formaldehyde Determination at Sub-PPM Level from Acetals by HPLC-DAD

ABSTRACT

Determination of formaldehyde at sub-ppm level as impurity in acetals using HPLC-DAD is described. Automated on-line precolumn derivatization reaction with 2,4 dinitrophenylhydrazine has been used. Breakdown rates of some industrial scale used acetals (Methylal, Ethylal) to formaldehyde by hydrolysis in aqueous media, according to pH, are described.     

MO-TMS Derivatives of Human Urinary Steroids for GC and GC-MS Studies

Abstract

Sterically hindered 17α-hydroxyl groups in steroids are silylated by trimethylsilylimidazole (TSIM) at 100° C. Decreasing rates of reaction were found for the following structures: 17α, 20β, 21-triol > 17α, 20α-diol > 17α, 20α, 21-triol. When methoxime derivatives are formed as the first step, HCL present in the excess of reagent catalyses the silylation reaction of hydroxyl groups. A simple procedure is described whereby methoxime (MO) derivatives are formed in 15 minutes at 60° C (the 11-one group does not react under these conditions) and persilylated compounds are then prepared in 2 hours at 100° C by reaction with trimethylsilylimidazole.