Determination of urinary glyoxal and methylglyoxal by high-performance liquid chromatography.

Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase high-performance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100-120 mOsm/kg H2O) under acidic conditions (pH 4.5) at 60 degrees C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within- and between-day relative standard deviations of less than 5%. Urinary levels (mean +/- standard deviation, n = 16) of glyoxal and methylglyoxal in healthy subjects were 4.7 +/- 1.35 microg/mg creatinine, 2.2 +/- 0.65 microg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.     

Quantitation of serum carotenoid concentrations in healthy inhabitants by high-performance liquid chromatography

Serum carotenoid concentrations in healthy subjects by age and sex were investigated, as determined by two high-performance liquid chromatography (HPLC) methods. Serum 'carotene' concentrations were 0.655 +/- 0.499 mumol/l 35.2 +/- 26.8 micrograms/dl) for 227 males, and 1,395 +/- 0.780 mumol/l (74.9 +/- 41.9 micrograms/dl) for 370 females. In addition, serum concentrations of alpha-carotene, beta-carotene, and lycopene were obtained from another sample included 356 males and 567 females living in the same area. alpha-Carotene for 356 males was 0.085 +/- 0.096 mumol/l (4.6 +/- 5.2 micrograms/dl), beta-carotene was 0.555 +/- 0.491 mumol/l (29.8 +/- 26.4 micrograms/dl), and lycopene was 0.372 +/- 0.273 mumol/l (20.0 +/- 14.7 micrograms/dl), respectively. Serum concentrations of alpha-carotene, beta-carotene as well as concentrations of lycopene in these inhabitants were significantly higher for females than for males (p less than 0.001).     

Assay of urinary free fucose by fluorescence labeling and high-performance liquid chromatography.

The concentrations of free fucose and other sugars in urine of cancer patients and healthy subjects were analyzed by high-performance liquid chromatography. After the urine samples were dried, we coupled the sugars in the residue with 2-aminopyridine to be detected as fluorescent derivatives. We then analyzed the pyridylamino derivatives of the sugars with an anion-exchange column and borate buffer. The difference between cancer patients and healthy subjects for the mean concentrations of fucose corrected for creatinine was significant (P less than 0.001). We checked the relationship between the concentrations of other sugars and the presence of cancer. This method is highly sensitive, and neither a cleanup procedure before labeling nor purification before injection into the column is needed. Not only fucose but also other sugars can be detected simultaneously, so this method should be useful for studying any changes in sugars in urine in various diseases.     

Differences in urinary albumin detected by four immunoassays and high-performance liquid chromatography

OBJECTIVES: To compare the analysis of urinary albumin from diabetic patients by four conventional immunoassays including radioimmunoassay (RIA), immunonephelometry (IN), and two different methods of immunoturbidimetry (IT), as well as by high-performance liquid chromatography (HPLC). DESIGN AND METHODS: Urines were collected over a 24-h period and stored at -20 degrees C until assay. Urinary albumin concentration was determined by an in-house RIA, by IN using a Beckman Array Analyser with reagents from Beckman Diagnostics (Sydney, Australia), by IT using a Dade-Behring Turbitimer with reagents from Dade-Behring (Marburg, Germany), by IT using a Dade-Behring Dimension R x L Chemistry Analyser with reagents from DiaSorin (Stillwater, OK, USA), and by HPLC using a Zorbax Bio series preparative GF-250 column. Regression lines were calculated using a least squares method to determine the correlation between the assays studied. Bland-Altman bias plots including limits of agreement were also calculated. RESULTS: The correlation coefficients calculated were high (>0.85) indicating a strong linear relationship between all assays studied. The slopes calculated for the comparisons demonstrate that each assay can vary from one another (up to threefold) and have a slope significantly different from an ideal slope of 1 (P < 0.001). These results were confirmed by Bland-Altman bias plots and calculation of the limits of agreement that were all large. CONCLUSIONS: At this time, there is no global standard by which urinary albumin assays may be standardized. This study suggests the need for such standards.     

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).     

Serum gentamicin assay by high-performance liquid chromatography.

We describe a high-performance liquid chromatographic method for the quantitative determination of gentamicin in serum. The antibiotic was separated from serum by passage through a silicic acid column, derivatized with o-phthalaldehyde, and eluted with ethanol. The derivatized gentamicin was then separated into all three of its major components by reversed-phase chromatography and quantified by fluorometry. Concentrations in serum as low as 0.5 mg of gentamicin per liter could be accurately determined. A standard curve showed a linear response for serum containing gentamicin at concentrations ranging from 0 to 20 mg/liter. Tobramycin, amikacin, ampicillin, penicillin G, methicillin, carbenicillin, chloramphenicol, clindamycin, and cephalothin did not interfere with the gentamicin assay. Comparison with an accepted microbiological assay yielded a correlation coefficient of 0.99. This chemical assay is rapid (less than 30 min), sensitive, accurate, specific, and appears to be applicable to other aminoglycosides.     

Measurement of adrenal corticosteroids by high-performance liquid chromatography.

OBJECTIVE. To develop a high-performance liquid chromatography (HPLC) method for simultaneous measurement of adrenal corticosteroids important for the diagnosis of congenital adrenal hyperplasia. DESIGN. Blind comparison using convenience samples. SETTING. Research laboratory at the Department of Medical Technology, Bowling Green State University. PATIENTS. Referred samples for cortisol, 11-deoxycortisol (S), and 17-hydroxyprogesterone (OHP). Stimulation condition unknown. INTERVENTION. Venous blood collected, processed to serum, and frozen. Adrenal steroids measured by radioimmunoassay (RIA) and by HPLC. MAIN OUTCOME MEASURES. Linearity, analytic sensitivity, accuracy, and precision were evaluated using recovery, coefficient of variation (c.v.), correlation coefficient (r), Student's t test, F test, and linear regression. RESULTS. Calibration curves were linear to at least 50 mumol/L, and the low-end sensitivity was 10 nmol/L. Analytic recovery ranged from 89.8% to 103.9%. The CV was below 7.5% (n = 10) for all three steroids. Comparison to RIA yielded r values of 0.909 (n = 25), 0.932 (n = 10), and 0.983 (n = 10) for cortisol, S, and OHP, respectively. Student's t-test results were 1.13 (p = 0.27), 0.10 (p = 0.93), and 0.48 (p = 0.64) for cortisol, S, and OHP, respectively. F-test results were 1.14 (p = 0.75), 1.37 (p = 0.66), 2.39 (p = 0.21) for cortisol, S, and OHP, respectively. Results for cortisol showed a negative bias of 97.0 nmol/L (3 mug/dL). Linear regression analysis revealed proportional errors of -15.0% -7.0%, and +9.0%, for cortisol, S, and OHP, respectively. CONCLUSION.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of ciprofloxacin in body fluids by high-pressure liquid chromatography.

We describe a reverse-phase high-pressure liquid chromatography method for the quantitation of a new quinoline carboxylic acid antimicrobial agent, ciprofloxacin (Bay o 9867). This assay utilizes the intrinsic fluorescence of ciprofloxacin for primary detection but employs UV absorption as a secondary detection system. Mobile phases contained methanol and phosphate buffer and used a common C18 mu Bondapak column. A single precipitation step of a 50-microliter specimen was the only sample preparation necessary. The assay is linear from 2,000 to 10 ng/ml and sensitive to 5 ng/ml. The mean recovery of ciprofloxacin from serum was 105.7%. The coefficient of variation was less than or equal to 3.1% for same-day precision and less than or equal to 6.3% for assay-to-assay precision. Because the assay requires only small specimen volumes and minimal sample preparation and because of its defined characteristics, this assay would be ideal for clinical trials and pharmacokinetics studies of ciprofloxacin.     

Apolipoprotein E haplotyping by denaturing high-performance liquid chromatography.

The apolipoprotein E ( APOE ) gene in humans contains two single-base polymorphisms in exon 4, which result in three common alleles, conventionally named epsilon2 , epsilon3 and epsilon4 . Numerous studies have shown an important association between the epsilon4 variant and an increased risk of Alzheimer's disease; other data suggest a possible linkage of APOE genetic heterogeneity with lipid profile and an increased risk of atherothrombotic stroke and coronary heart disease. APOE genotyping is therefore an increasingly common assay in laboratory medicine. The most widely used technique for APOE genotyping is based upon restriction isotyping, i.e., amplification of the fragment of exon 4 containing the most common sequence variations, followed by enzymatic digestion of the amplicon and fragments analysis by gel electrophoresis. We developed a novel, reliable and fast method that exploits the sensitivity and specificity of denaturing high-performance liquid chromatography in detecting single-nucleotide polymorphisms. We show that, in most cases, with a single chromatographic separation it is possible to correctly identify the six different APOE allelic patterns, without any manipulation after the polymerase chain reaction amplification. When compared to restriction isotyping, our method is much faster, less labor intensive and similarly inexpensive.     

Determination of quinidine by high-performance liquid chromatography

Quinidine in serum was measured at 330 nm, after protein precipitation, with an alkyl phenyl reversed-phase chromatographic column by peak-height determination (external standards.) Sensitivity was 0.3 mg/liter, with linear response to at least 10 mg/liter serum. Interassay precision, measured on 20 consecutive days, gave a CV of 8.2% at 2 mg/liter of serum and 5.1% at 5 mg/liter. Quinine and primaquine are not separable from quinidine under the assay conditions, and dihydroquinidine and chloridazepoxide are only partly resolved. No assay interference was encountered with a series of control serum samples obtained from patients with various diseases, who were being treated with various drugs other than quinidine, except in one serum sample with a high bilirubin concentration (300 mg/liter). If such an assay interference is present, an alkaline extraction of quinidine before analysis has to be used. Results by our assay and those by a single-extraction fluorescence method correlated poorly (r=0.87), and the fluorescence assay gave 50% +/- 7% (SEM) higher values than our method, due to chromatographic separation of a major fluorescent non-phenolic metabolite of quinidine, which can also be measured by our assay.     

Quantitation of hemoglobin A1a+b and hemoglobin A1c by automated "high-performance" liquid chromatography.

The glycosylated hemoglobins A1a+b and A1c have been rapidly and precisely quantitated in 5-micro L samples of human blood hemolysate (approximately 240 micrograms of hemoglobin) by cation-exchange column chromatography. Total chromatographic is 22.0 min. Proportions of Hb A1c range from 3.85 to 6.71% in normal individuals and from 4.23 to 19.90% in diabetic subjects. Within-day variation was 1.58 and 1.10% for mean Hb A1c proportions of 4.92 and 10.32%, respectively. Hb A1c and Hb A1 are stable in hemolysates stored at 4 degrees C for as long as seven days, and indefinitely under liquid nitrogen.     

Determination of carbamylated hemoglobin by high-performance liquid chromatography

We have developed an HPLC method for measuring carbamylated hemoglobin (CarHb), based on the quantification of valine hydantoin formed from the released NH2-terminal carbamyl valine residue after acid hydrolysis of hemoglobin. In uremia, CarHb is produced by nonenzymatic post-translational modification of the terminal amino group of hemoglobin monomers by isocyanic acid, derived from the spontaneous dissociation of urea. We measured CarHb in 25 nonuremic control subjects, 24 nonuremic diabetic subjects, and 30 patients with stable chronic renal failure. There was no significant difference between the controls and diabetic patients, their mean (SD) CarHb values being 41 (11.5) and 38 (10.8) micrograms of carbamyl valine per gram of hemoglobin (microgram CV/gHb), respectively. Mean (SD) CarHb values in the uremic patients were much greater, 164 (87.7) microgram CV/gHb. There was significant correlation between the concentrations of CarHb and plasma urea in the uremic subjects. Thus CarHb provides a urea-derived index of chronic uremia.     

高效液相色谱法检测氨甲酰血红蛋白

目的建立一种测定氨甲酰血红蛋白的高效液相色谱法。方法将血红蛋白经酸水解后，释放出氨甲酸缬氨酸，并进一步转化成缬氨酸乙内酰脲后进行定量分析。结果线性范围为０～８０ｍｇ／Ｌ，最低检测量为１４μｇＣＶ／ｇＨｂ平均回收率为７４５７％，批内变异系数（ＣＶ）为８５％，批间ＣＶ为１０９％。并对３０例正常人进行测定。结论该方法快速、灵敏、特异、实用，适于临床常规和研究应用。     

Analysis of 8-methoxypsoralen by high-performance liquid chromatography

We report a simple and rapid procedure for assaying 8-methoxypsoralen (8-MOP) in plasma by high-performance liquid chromatography (HPLC). The standard curve for the assay is linear for 8-MOP from 15 to 500 micrograms/L (y = 0.002x-0.01, r = 0.99) with a lower limit of detection of 1.5 micrograms/L. Intra-assay precision (CV) was 6.0% at the 100 micrograms/L concentration and 10.0% at 50 micrograms/L (n = 30 each). Interassay precision was 6.4% at 100 micrograms/L and 7.0% at 50 micrograms/L (n = 50 each). Extraction recovery of 8-MOP was 98%. Common antiarrhythmics, sedatives, and hypnotics were found not to interfere.     

A simple quantitative assay for urinary adenosine using column-switching high-performance liquid chromatography

Adenosine is a physiologically active molecule produced locally in many sites of the body to regulate various cell functions. Measurement of levels of the factor in organs and biological fluids provides clues to its role and we reported an accurate quantitative high-performance liquid chromatography method for urinary adenosine requiring no preliminary sample preparation, other than filtration. Analyses were performed isocratically with a reversed-phase and a molecular exclusion columns connected by a column switch. Each sample was analyzed automatically in 35 min. Linearity could be verified up to 1,000 micromol/L (r = 0.999) and recovery of adenosine was 94.6-98.0%. The coefficients of variation (CV) were established to be 0.56-1.32%, intra-assay, and 1.61-4.67%, inter-assay. Based on analyses of healthy individuals at different ages, we are here able to provide age-related values, infants (1.51 +/- 0.71 micromol/mmol creatinine) and children (1.06 +/- 0.36 and 0.83 +/- 0.27 micromol/mmol creatinine; aged 1-5 and 6-10 years), excreting significantly higher amounts of adenosine than adults (0.44 +/- 0.08 micromol/mmol creatinine). We also measured urinary adenosine from patients suffering from metabolic disease or severe respiratory failure and found that unfavorable pathophysiologic conditions are associated with appreciable elevation of adenosine.     

Quantitation of glucocorticoids in blood serum by high-efficiency liquid chromatography

The paper presents a method of quantitation of endogenous and exogenous glucocorticoids by high-efficiency liquid chromatography. The developed technique is implemented on the basis of Russian equipment and Russian reagents (except for etalons). A "Milichrom" Russian chromatograph is convenient and safe in operation; as for the detection limit, it is not inferior to its foreign analogues. The suggested technique is simple, safe and can be implemented at city and district patient-care facilities.     

Quantitation of bilirubin conjugates with high-performance liquid chromatography in patients with low total serum bilirubin levels

Bilirubin mono- and diconjugates were determined by alkaline methanolysis and high-performance liquid chromatography (HPLC) in serum from patients with metastatic liver disease and liver cirrhosis. Conjugates could be detected and quantitated at normal or low total bilirubin levels. Comparison with serum alkaline phosphatase activity revealed that in cirrhosis bilirubin conjugates were sometimes detectable at normal or slightly elevated alkaline phosphatase activities. In patients with metastatic liver disease alkaline phosphatase activity was a more sensitive indicator. In normal controls and in patients with Gilbert's syndrome no bilirubin conjugates were detected whereas serum of patients with haemolysis contained conjugated bilirubin. Therefore HPLC appears to be an excellent method to diagnose Gilbert's syndrome. In liver cirrhosis HPLC is a useful liver function test.     

Assay of fluconazole by high-performance liquid chromatography with a mixed-phase column.

A mixed-phase liquid chromatographic column was used to assay fluconazole in plasma, serum, and cerebrospinal fluid. The assay was linear from 0.2 to 20 micrograms/ml, with an average coefficient of variation of less than 5%. The partitioning of the drug between serum and cerebrospinal fluid was determined for 34 patients. The method was demonstrated to be suitable for both pharmacokinetic studies and monitoring of patients receiving treatment with this antifungal agent.     

Measurement of red blood cell adenosine nucleotides by high-performance liquid chromatography.

A new method for measuring ATP in stored red blood cells using high-performance liquid chromatography was compared with an established enzymatic method. The new method is based upon isocratic reverse phase chromatography using a polyvinyl alcohol gel stationary phase. The chromatograms produce quantitative results for ADP, AMP, and other nucleotides, and can be used to determine adenylate energy charge. The correlation coefficient between the two methods was 0.91, and mean ATP was 3.0 mumol/g Hb for both methods. Tests of hypothesis for mean and variance were not significant. The method is recommended as a means to study the relationships between poststorage red blood cell ATP, adenylate energy charge, total adenylates, and posttransfusion erythrocyte survival.