Evaluation of 2-iminoimidazolidin-4-one and thymine as respective internal standards for normal-phase and reversed-phase high-performance liquid chromatographic determination of creatinine in human serum

We evaluated two internal standards for HPLC determination of creatinine in human serum after ultrafiltration: 2-iminoimidazolidin-4-one for normal-phase HPLC on aluminium oxide, and thymine for C₁₈ reversed-phase HPLC. Detection of 2-iminoimidazolidin-4-one was done at the same wavelength as that used for creatinine, i.e. 240 nm. For thymine, the wavelength was switched to 280 nm. The suitability of the selected compounds to serve as an internal standard in the described measurement procedures, including ultrafiltration of serum, was evaluated from the precision and accuracy obtained. The method based on normal-phase HPLC with 2-iminoimidazolidin-4-one showed an imprecision expressed as R.S.D. ranging from 0.8 to 3.4% (mean: 2.1%) and an inaccuracy, calculated from the deviations from target values determined by isotope-dilution gas chromatography-mass spectrometry, ranging from −1.3 to +1.8% (mean: +0.4%). For the reversed-phase HPLC procedure with thymine, the imprecision ranged from 0.3 to 1.3% (mean: 1.0%) and the inaccuracy from +0.1 to 3.9% (mean: +1.7%). The occasional observation of interferences with 2-iminoimidazolidin-4-one limited the application of the normal-phase method to a certain extent.     

Measurement of creatinine by Jaffe's reaction--determination of concentration of sodium hydroxide required for maximum color development in standard,urine and protein free filtrate of serum

Creatinine in serum or urine is determined by Jaffe's reaction where creatinine produces quantitatively an orange color with picric acid in alkaline medium. After allowing an incubation time of 15 min at room temperature for color development the color is measured at 520 nm. Without taking into consideration the acidic nature of standard, protein free filtrate (PFF) of serum and urine, 1% picric acid and 0.75N NaOH are used in this reaction for color development in standard, PFF of serum and urine. An investigation was thought to be necessary to determine the optimum alkali concentration required in standard, PFF of serum and urine. The results show that 0.25, 0.75 and 1N NaOH give maximum color in urine, standard and PFF of serum respectively. A standard solution of creatinine is prepared in 0.1N HCl and the PFF of serum is obtained by addition of fresh tungstic acid. Alkali is consumed to neutralise the acids in both these cases. For urine creatinine measurement, a direct diluted urine sample is used. The difference in the requirement of NaOH is conceivable. The routine use of 0.75N NaOH irrespective of the nature of specimen as is done in all biochemical laboratories, for creatinine measurement needs modification in the light of this investigation.     

Assay of inorganic sulfate in biologic fluids by nonsuppressed (single-column) ion chromatography

An assay using nonsuppressed (single-column) anion chromatography was developed to determine the concentration of inorganic sulfate in biologic fluids. A conventional HPLC system with an anion-exchange column and conductimetric detector interfaced with an automatic injector and integrator was used. The mobile phase for the chromatography of urine and serum samples is 4 mM potassium hydrogen phthalate, pH 4.5, and potassium iodide is used as the internal standard. For cerebrospinal fluid samples, the mobile phase is modified by addition of 10% of a 4 mM phthalic acid solution. Results of the HPLC assay were found to correlate well (r = 0.991 and 0.999) with those of two commonly used spectrophotometric methods for urine and serum inorganic sulfate determinations. However, the concentrations determined by ion chromatography were 2.5 to 10% lower, possibly due to less assay interference by other substances following chromatographic separation of sulfate. Anion chromatography using a single-column system is a convenient and relatively inexpensive method with sufficient sensitivity for the determination of inorganic sulfate concentrations in urine, serum, and cerebrospinal fluid.     

Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography with ultraviolet detection

A combination of two stereoselective assays was developed using column-switching HPLC with electrochemical detection for the determination of free (unconjugated) silibinin and RP-HPLC with UV detection for the measurement of total (free and conjugated) silibinin in human plasma. After extraction of free silibinin and the internal standard hesperetin with diethyl ether the compounds were pre-separated on a RP-CN column. A cut fraction of eluate containing the analytes was then transferred to the RP-18 main column by means of a switching valve for final separation of the compounds. The limit of quantification with electrochemical detection for free silibinin was 0.25 ng/ml per diastereomer. For the determination of total silibinin diastereomers all conjugates were cleaved enzymatically using β-glucuronidase/arylsulfatase at pH 5.6 followed by extraction with diethyl ether of the pH 8.5 alkalized solution. Separation of the diastereomers and of the internal standard naringenin was achieved on a RP-18 column. The limit of quantification with UV detection at 288 nm for total silibinin was 5 ng/ml per diastereomer. Both assays were successfully applied to the stereospecific analysis of silibinin in plasma samples from a pharmacokinetic study of silymarin in human volunteers.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.     

Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples

A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.     

Lipase-catalyzed synthesis of partial glyceride

Summary Mucor miehei (IM 20) and Candida antarctica (SP 382) lipases were used for esterification of free fatty acids in the absence of organic solvent or transesterification of fatty acid methyl esters in hexane with isopropylidene glycerols. Acid catalyzed cleavage of the isopropylidene groups resulted in the formation of monoacyl glycerol (MAG) and diacyl glycerol (DAG). Both oleic (18:1 n-9) and eicosapentaenoic acid, EPA (20:5 n-3) were successfully incorporated into glycerides. Total acyl donor conversion ranged from 46.9 – 96.9% with MAG content of up to 88.5%.     

Colorimetric Measurement of Triglycerides Cannot Provide an Accurate Measure of Stored Fat Content in Drosophila

Drosophila melanogaster has recently emerged as a useful model system in which to study the genetic basis of regulation of fat storage. One of the most frequently used methods for evaluating the levels of stored fat (triglycerides) in flies is a coupled colorimetric assay available as a kit from several manufacturers. This is an aqueous-based enzymatic assay that is normally used for measurement of mammalian serum triglycerides, which are present in soluble lipoprotein complexes. In this short communication, we show that coupled colorimetric assay kits cannot accurately measure stored triglycerides in Drosophila. First, they fail to give accurate readings when tested on insoluble triglyceride mixtures with compositions like that of stored fat, or on fat extracted from flies with organic solvents. This is probably due to an inability of the lipase used in the kits to efficiently cleave off the glycerol head group from fat molecules in insoluble samples. Second, the measured final products of the kits are quinoneimines, which absorb visible light in the same wavelength range as Drosophila eye pigments. Thus, when extracts from crushed flies are assayed, much of the measured signal is actually due to eye pigments. Finally, the lipoprotein lipases used in colorimetric assays also cleave non-fat glycerides. The glycerol backbones liberated from all classes of glycerides are measured through the remaining reactions in the assay. As a consequence, when these assay kits are used to evaluate tissue extracts, the observed signal actually represents the amount of free glycerols together with all types of glycerides. For these reasons, findings obtained through use of coupled colorimetric assays on Drosophila samples must be interpreted with caution. We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.     

基因工程菌高密度发酵液中碳源物质甘油的定量检测及其浓度的优化控制

比较了高碘酸氧化法,铜离子络合法、高压液相色谱法和甘油激酶法四种不同的方法定量测定重组工程菌ＹＫ５３７／ＰＳＢ－ＴＫ高密度发酵液中甘油的浓度,发现甘油激酶法可以排除发酵液中其他物质的干扰,精确测定甘油的含量。在此基础上对发酵培养中甘油的浓度进行了优化,结果表明,甘油浓度控制在较低的水平有利于菌体密度的提高。在５Ｌ自动发酵罐中,控制甘油浓度在５ｇ／Ｌ左右,经２０ｈ的培养,最终细菌密度达到１２０ＯＤ６     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

Radioimmunoassay of unconjugated and total serum estetrol using a 125I-iodinated tracer

A radioimmunossay (RIA) for the measurement of both unconjugated and total serum estetrol has been developed, using an antiserum to an E4-3-conjugate and a 125I-radioiodinated E4 tracer. Assay of dried ethyl ether extracts was used for the determination of unconjugated E4, while a direct measurement of unextracted hydrolyzed serum in the presence of 0.3% 8-anilino-1-naphthalene sulphonic acid (ANS) proved adequate for total E4. Assay reliability was evaluated and the procedure standardized through a series of tests aimed at assessing accuracy, sensitivity and precision. No steroidal interference was found to practically affect the assay (0.3% estriol cross-reactivity), nor were solvent and sample blanks observed in the case of unconjugated E4. For total E4 assay, the sample blank effects were acceptably overcome by using hydrolyzed male serum and 0.3% ANS, as a standard diluent. An interassay variability amounting to approximately 10 and 6% resulted for unconjugated E4 and total E4 RIA, respectively. A number of serum samples (285 for unconjugated E4, 147 for total E4) randomly collected throughout normal pregnancy were assayed. The unconjugated E4 levels at 15th week and at term were 62.7 +/- 22.6 and 766.5 +/- 208.2 (SD) pg/ml, respectively. Total E4 was about 6--7 times higher than the levels of free E4 and increased 7 times from the 15th week to term.     

Radiometric assays for glycerol, glucose, and glycogen

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.     

Urinary 5-HIAA measurement using automated on-line solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC-MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d(2)) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d(2)) were retained on and eluted from the SPE cartridges in high yields (81.5-98.0%). Absolute recovery was 96.5-99.6%. Intra-assay (n=20) and inter-assay (n=20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n=78) with known or suspected metastatic carcinoid tumors, results obtained by XLC-MS/MS were highly correlated (R(2)=0.99) with the routinely used fluorometric method. This XLC-MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.     

Rapid microdetermination of fatty acids in biological materials by gas-liquid chromatography

Total and free fatty acids in general ranging from lauric to nervonic acid were separated and quantitated based on an internal standard method as methyl esters by “on column” methylation with trimethyl-(α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) in a gas chromatographic system. This study represents an application of a method published by MacGee and Allen and a change to an internal standard technique. For the determination of the total fatty acids the sampls were saponified with KOH-CH₃OH, acidified with H₂PO₄, and then the fatty acids were extracted into hexane. An aliquot of the hexane extract was then extracted with TMTFTH and chromatographed. For determination of free fatty acids the sample was acidified with H₃PO₄, immediately extracted with hexane and processed as described earlier. The relative standard deviation of 1.4 to 4.2% illustrates the precision of the method and the recovery of the fatty acids ranged from 88.5 to 100.5%. This method was applied to the determination of fecal fatty acids in conjunction with an interdepartmental study on “High protein diet in colon cancer” at the University of Missouri. In addition, the applicability of the analytical procedure (with small modifications) was shown for a wide variety of biological materials (serum, milk, skin tissue, fungal spores, food homogenates, beef tissues, and tumor cell cultures). The analyses were performed on different gas chromatographs by different analysts.     

Determination of free and total 7-hydroxycoumarin in urine and serum by capillary electrophoresis

A new method for the rapid determination of 7-hydroxycoumarin, the predominant metabolite of coumarin in humans, was developed for analysis in urine and serum, based on separation by capillary electrophoresis, with UV detection at 210 nm. The linear detection range for 7-hydroxycoumarin was 0–50 μg/ml while the limit of quantitation was 1 μg/ml. An internal standard, 3-(α-acetonylbenzyl)-4-hydroxycoumarin, was utilised for the determination of free 7-hydroxycoumarin, but it was found not to be suitable in the analysis of total 7-hydroxycoumarin present. Urine from two volunteers, who had been administered coumarin, was analysed by both capillary electrophoresis and by HPLC. The results from the two methods were compared and contrasted. The CE method was found to decrease the analysis time in comparison to HPLC analysis, with results available after 1.5 min as compared to 12 min with HPLC. There was no statistical difference between the results determined by either method.     

Measurement of carnitine precursors, ε-trimethyllysine and γ-butyrobetaine in human serum by tandem mass spectrometry

Methods using tandem mass spectrometry for measurement of ε-trimethyllysine and γ-butyrobetaine in human serum are described. Precursor ion scan analysis of a methylated sample was applied for γ-butyrobetaine measurement. However, for ε-trimethyllysine measurement, homoarginine interfered with the methylated sample during precursor ion scan analysis. To overcome this interference, the sample was propylated and acetylated prior to precursor ion scan analysis. The obtained values resembled those obtained by enzymatic or HPLC measurement. Using tandem mass spectrometry, all members of the carnitine family, free carnitine, acylcarnitines, γ-butyrobetaine, ε-trimethyllysine can be analyzed in 0.1 ml of serum. Thus, the proposed method appears to be suitable for clinical application, especially in the pediatric field.     

Evidence for the presence of a pool of glycerides with a rapid rate of turnover in brown fat from newborn rabbits

1. The specific radioactivity of [(14)C]glycerol released during the incubation of brown fat with [(14)C]glucose is much greater than that of the tissue lipid glycerol. 2. From a study of the release of [(14)C]glycerol from pre-labelled brown fat, it is concluded that the tissue contains a pool of glycerides with a higher rate of turnover than those in the main lipid store. 3. This pool contains newly synthesized glycerides, has a half-life of 25-30min and supplies about 25% of the glycerol liberated by brown fat. 4. Thus, a significant fraction of the total (14)C incorporated from glucose into brown-fat lipids is released as [(14)C]glycerol during an incubation.     

Flow injection analysis-based methodology for automatic on-line monitoring and quality control for biodiesel production

An automated on-line approach based on determination of free and bound glycerol was here proposed to monitor biodiesel production. The method was based on liquid-liquid extraction of glycerol from the biodiesel to an aqueous ethanolic phase in which glycerol is oxidized to formaldehyde with meta periodate with subsequent reaction with acetylacetone. The reaction product was photometrically measured at 410 nm. Free and bound glycerol were differentiated by glycerides hydrolysis with potassium ethylate. The experimental set-up consisted of a flow-injection manifold for liquid-liquid extraction without phase separation and iterative change of the flow direction that enabled: (a) filling the flow manifold with a meta periodate-acetylacetone acceptor phase; (b) sampling of small amounts (microl) from the reactor; (c) determination of free glycerol by extraction from biodiesel to the aqueous phase with simultaneous oxidation-reaction with acetylacetone in the acceptor phase; (d) continuous monitoring of the aqueous phase by passage through a photometric detector; (e) filling the flow manifold with a potassium ethylate-meta periodate-acetylacetone new acceptor phase; (d) repetition of steps b-to-d to determine total glycerol after saponification of the bound glycerol by potassium ethylate; and (f) determination of bound glycerol by difference between the second and first analyses. The results showed that the proposed automated on-line method is a suitable option in routine analysis during biodiesel production.     

The effect of starvation on the incorporation of palmitate into glycerides and phospholipids of rat liver homogenates

1. Glyceride biosynthesis from glycerol phosphate and [1-(14)C]palmitate was studied in liver homogenates of rats that were fed ad libitum or starved for 36-40hr. The changes in enzyme activity were related to total DNA content or total liver homogenate as these were found to be equivalent and to be the most meaningful parameters. 2. In liver homogenates from fed rats, labelled palmitate was incorporated mainly into phosphatidate (58% of the total incorporation into lipids), diglycerides (25%) and triglycerides (16%), whereas monoglycerides, cholesterol esters and phospholipids other than phosphatidate were labelled only to a small extent. Addition of particle-free supernatant to full homogenates increased the total incorporation of palmitate by 45% and the pattern of incorporation altered to 53% incorporated into triglycerides, 24% into diglycerides and 17% into phosphatidate. This result suggested that, in liver homogenates, phosphatidate phosphohydrolase (EC 3.1.3.4) may be rate-limiting in the biosynthesis of glycerides via the glycerol phosphate pathway. 3. Upon starvation, the amount of palmitate incorporated per liver into total phospholipids plus glycerides was decreased to between 68% and 75% of that observed with fed animals. In homogenates from fed animals 41-44% of the labelled phospholipids plus glycerides was in glycerides; this value increased to between 63% and 75% with starved rats. Of the palmitate incorporated into total phospholipids, between 85% and 86% was found in phosphatidate, independent of the nutritional state of the animal. The ratio of palmitate incorporated into triglycerides/diglycerides rose from 0.7, obtained with fed rats, to 1.0 with starved animals. 4. These results indicate that starvation caused a decrease in the activity (per total liver) of acyl-CoA-glycerol phosphate acyltransferase(s) (EC 2.3.1.15) and an increase in the activity of acyl-CoA-diglyceride acyltransferase (EC 2.3.1.20). The largest change, however, seemed to be related to the increased activity of the phosphatidate phosphohydrolase in the particle-free supernatant. 5. The latter enzyme was assayed in the particle-free supernatant with membrane-bound phosphatidate as substrate. In starvation, the activity per total liver was increased to between 130% and 190% and the specific activity to between 180% and 320% of the values for fed rats.     

Validated liquid chromatographic determination of 5-fluorouracil in human plasma

A sensitive, reproducible, selective and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of 5-flurorouracil in plasma has been developed and validated using isocratic elution and UV detection. The method provides a selective quantifications of 5-flurorouracil without any interference of the endogenous uracil. The assay is performed after a double extraction of 5-flurorouracil and thymine (internal standard) from human plasma using ethyl acetate. The drug and the internal standard were eluted from a Genesis C(18) analytical column at ambient temperature with mobile phase consisting of methanol:water (10:90, v/v) adjusted to pH 3.2 with perchloric acid at a flow rate of 1.0 ml/min. The effluent was monitored with an ultraviolet detector at 260 nm. Quantification was achieved by the measurement of the peak-height ratios and the limit of quantification for 5-flurorouracil in plasma was 30 ng/ml. The retention times for 5-flurorouracil, uracil, and thymine were 4.5, 6.0, and 9.0, respectively. The intra-day coefficient of variation (CV) ranged from 1.35 to 4.53% at three different concentrations and the inter-day CVs varied from 1.29 to 4.98%. The relative and absolute recoveries varied from 96 to 101%. Stability tests showed that 5-flurorouracil is stable for at least 72 h in plasma after freezing. The simple method may permit the assessment of 5-flurorouracil plasma concentrations for pharmacokinetic studies in combination with clinical trials.