Simple high-performance liquid chromatographic analysis of phenol and p-cresol in urine and feces

Previous reports which present methods of analysis of phenol and p-cresol by HPLC are usually designed for the detection of these compounds in urine, can be complicated by the use of uncommon equipment or additional techniques such as steam distillation or derivatisation, or concentrate on the detection of phenol rather than p-cresol. In this paper we report a simple method suitable for the analysis of phenol and p-cresol in both urine and feces, based upon extraction into ether following acid hydrolysis and UV detection.     

Measurement of endogenous Na+,K+-ATPase inhibitors in human plasma and urine using high-performance liquid chromatography

This study was undertaken to assess endogenous Na+,K+-ATPase inhibitors in both plasma and urine in the same subjects. Samples were chromatographed on reverse-phase HPLC using an acetonitrile gradient and the eluent screened using Na+,K+-ATPase inhibition and cross-reaction with anti-digoxin antibodies. The donors were divided into inhibiting and non-inhibiting subjects using a previously described method, plasma action on ouabain binding and on Na+,K+-ATPase activity. Three Na+,K+-ATPase inhibitors (1P, 2P and 3P) were detectable in plasma; the antibodies cross-reaction of the peaks 2P and 3P were larger than that of peak 1P. The peaks 2P and 3P were significantly higher in inhibiting subjects as compared to non-inhibiting subjects. The 24-h urine is resolved into two peaks inhibiting Na+,K+-ATPase activity (1U and 2U). Peak 2U cross-reacted with anti-digoxin antibodies to a greater extent than peak 1U and is significantly larger in inhibiting subjects in terms of Na+,K+-ATPase inhibition. These data support the heterogeneity of human Na+,K+-ATPase inhibitor in both plasma and urine.     

Detection of Salmonella in feces by using Felix-01 bacteriophage and high-performance liquid chromatography

Utilizing the Felix-01 bacteriophage and high-performance liquid chromatographic technique, a method was devised making possible the detection of approximately 10⁶Salmonella typhimurium/g of human feces within 8–9 h of sample collection. Using overnight enrichment, as few as 10³S.typhimurium/g of feces were detectable.     

Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix

A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r2=0.999) was acceptable over the range of 10-5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or -20 degrees C.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.     

High-performance liquid chromatography determination of glyoxal,methylglyoxal, and diacetyl in urine using 4-methoxy-o-phenylenediamine as derivatizing reagent

Bioanalytical relevance of glyoxal (Go) and methylglyoxal (MGo) arises from their role as biomarkers of glycation processes and oxidative stress. The third compound of interest in this work is diacetyl (DMGo), a component of different food products and alcoholic beverages and one of the small α-ketoaldehydes previously reported in urine. The original idea for the determination of the above compounds by reversed phase high-performance liquid chromatography (HPLC) with fluorimetric detection was to use 4-methoxy-o-phenylenediamine (4MPD) as a derivatizing reagent and diethylglyoxal (DEGo) as internal standard. Acetonitrile was added to urine for matrix precipitation, and derivatization reaction was carried out in the diluted supernatant at neutral pH (40 °C, 4 h); after acidification, salt-induced phase separation enabled recovery of the obtained quinoxalines in the acetonitrile layer. The separation was achieved within 12 min using a C18 Kinetex column and gradient elution. The calibration detection limits for Go, MGo, and DMGo were 0.46, 0.39, and 0.28 μg/L, respectively. Within-day precision for real-world samples did not exceed 6%. Several urine samples from healthy volunteers, diabetic subjects, and juvenile swimmers were analyzed. The sensitivity of the procedure proposed here enabled detection of differences between analyte concentrations in urine from patients at different clinical or exposure-related conditions.     

蕲蛇酶在实验性动物的尿,粪排泄率和血浆蛋白结合率研究

用１３１Ｉ标记方法研究蕲蛇酶在大鼠尿、粪中排泄情况和与家兔血浆蛋白结合率,结果表明,其自大鼠尿中排泄率为结合量的３２．８±ｓ０．４８％,粪中排泄率为４．９６±ｓ０．９９％,与家兔血清蛋白结合率为３０．６３％。     

Whole blood and urine bioactive Hepcidin-25 determination using liquid chromatography mass spectrometry

Hepcidin is a small cysteine-rich signaling peptide that regulates blood serum iron concentrations [1–4]. Patients with chronic inflammation are known to have elevated levels of hepcidin in their blood and urine and often suffer from anemia as a result [5–10]. Measuring and quantifying the amount of active hepcidin in blood and urine can help to determine the cause and severity of the anemia thereby helping physicians determine the correct course of treatment [11–16]. We have developed a simple technique to isolate, chemically modify, and concentrate hepcidin from blood and urine coupled to high-pressure liquid chromatography mass spectrometry that can accurately and reproducibly measure and quantify the active hormone.     

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C₁₈ column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 ± 3.7% (n = 45) and 100 ± 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 μmol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

Measurement of plasma acetate kinetics using high-performance liquid chromatography.

Previous studies suggest that plasma acetate may be an important fuel in man, accounting for approximately 10% of energy expenditure. Available methods for the determination of plasma acetate kinetics are difficult and time consuming. We describe here a procedure for the determination of plasma acetate concentration and specific activity using automated high-performance liquid chromatography that is precise and sensitive and accommodates large numbers of samples. The procedure involves extraction from plasma with diethyl ether, derivatization with bromoacetophenone, and separation on a C-18 reversed-phase column. The specific activities of D-beta-hydroxybutyrate and lactate can also be determined. Acetate turnover was measured in four dogs and was similar to that previously reported in sheep and humans. Transport of [14C]acetate into red blood cells was negligible.     

Quantification of penicillin-G and procaine in equine urine and plasma using high-performance liquid chromatography

A rapid and sensitive method for the extraction and quantification of penicillin-G and procaine in horse urine and plasma samples has been successfully developed. The method involves the use of solid-phase extraction (SPE) for penicillin-G, liquid–liquid extraction (LLE) for procaine, and high-performance liquid chromatography (HPLC) for the quantification of penicillin-G and procaine. The new method described here has been successfully applied in the pharmacokinetic studies of procaine, penicillin-G and procaine–penicillin-G administrations in the horse.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Determination of codeine and metabolites in plasma and urine using ion-pair high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the simultaneous determination of codeine and seven metabolites is described. The samples are purified by reversed-phase solid-phase extraction. Codeine, norcodeine, codeine-6-glucuronide, norcodeine-6-glucuronide and morphine-3-glucoronide are measured with UV detection. Detection limits are 3 nmol/l (morphine-3-glucuronide) to 20 nmol/l (codeine). Morphine, normorphine and morphine-6-glucuronide are measured with electrochemical detection. Detection limits are 0.4 nmol/l (morphine-6-glucuronide) to 1.0 nmol/l (normorphine). Correlation coefficients better than 0.998 are normally obtained for all compounds. The method was applied to the determination of the kinetics of codeine and its metabolites in plasma and urine samples from healthy volunteers.     

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.