Total urinary estriol determined by "on-line" liquid chromatography with ultraviolet detection

We report a fully automated method for the specific assessment of total estriol in urine. Acid-hydrolyzed urine is concentrated on a short reversed-phase column and prepurified with methanolic Tris buffer, the pH of the buffer increasing from 7 to 11. The anionic form of the estriol molecule is eluted with methanolic NaOH. By acidification in a mixing chamber, estriol in its neutral form is focused on the top of a second short reversed-phase column, effectively chromatographed on an analytical column, and quantified by ultraviolet absorbance at 278 nm. Losses of estriol throughout the total procedure are negligible and thus external calibration is feasible for quantification. Analytical recoveries for estriol-supplemented urines ranged from 98.3 to 105%. Replicate analyses of a urine containing 68 mumol of estriol per liter gave a CV of 2.76%. As little estriol as 2 mumol/L can be detected. Results from pregnancy urines correlated well with those of radioimmunoassay. The method is especially suited for clinical emergencies in a routine laboratory.     

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.     

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.     

Urinary free methyldopa determined by reversed-phase high-performance liquid chromatography

We describe a chromatographic method, in which 3,4-dihydroxybenzylamine is used as the internal standard, for determining free methyldopa in human urine. The drug was adsorbed onto alumina, eluted, and the eluate directly injected onto a reversed-phase column (octadecyl-bonded silica stationary phase), with dilute acetate buffer (pH 2.7) as the mobile phase and ultraviolet detection at 280 nm facilitated. Methyldopa is well separated from other urinary biogenic amines present in the alumina extract, and other commonly used antihypertensives and diuretics do not interfere with the analysis. The sensitivity of the method is adequate to quantify 8.0 mg of methyldopa per liter in 30 ml of sample; the lower limit of detection is 25 ng. Analytical recovery for methyldopa varied from 95 to 102% with within-run and day-to-day coefficients of variation of 2.7 (n = 10) and 3.8% (n = 5), respectively. This procedure is readily adaptable for use in studies of the pharmacokinetics of methyldopa and to routine clinical laboratory use.     

Highly specific, simple and rapid method for the determination of malondialdehyde in blood using high-performance liquid chromatography.

A novel, highly specific, simple and rapid method for the determination of malondialdehyde (MDA), the routinely used marker for free radical generation in body fluids has been developed and evaluated. Serum samples from 30 healthy volunteers in heparin and 1,4-dithiothreitol-containing tubes stored at -80 degrees C were analyzed. The MDA-thiobarbituric acid complex was separated from interfering substances using HPLC. For the separation, reverse phase column MAC (4 x 250 mm, Biospher SI 120 PSI C18, particle size 7 microm) was used. The mixture of methanol and 8.3 mmol/l phosphate buffer, pH= 7.2, (35:65, v/v) was used as mobile phase. The volume of serum samples injected on the column was 50 microl. The analyte was detected at 532 nm. Retention time of MDA-thiobarbituric acid complex was 4.9+/-0.1 min at the flow rate 0.7 ml/min. Excellent linearity was achieved. The intra- and interassay coefficient of variation was 7.3% and 8.8%, respectively. The recovery was 95.6% and the detection limit was 0.1 micromol/l. The validity of this method was proved by comparison with the spectrophotometric determination of MDA-thiobarbituric acid complex by the method of Yagi at three different wavelengths (485, 532 and 560 nm) with Allen's correction.     

Selective determination of hydroxyproline in urine by high-performance liquid chromatography using precolumn derivatization

A method to measure total hydroxyproline in human urine was developed. Primary amino acids were derivatized with ortho-phthaldialdehyde, followed by derivatization of imino acids with 9-fluorenylmethyl chloroformate. The fluorescent 9-fluorenylmethyl chloroformate derivatives were separated by reversed phase high-performance liquid chromatography. 3,4-Dehydroproline was used as internal standard. Calibration curves for hydroxyproline and internal standard were linear from 1 to 200 pmol injected. Both within- and between-run precision were below 3.2%. Analytical recovery of hydroxyproline added to urine samples was 99.2 +/- 2.6%. Values for excreted hydroxyproline were determined by analysis of urine samples from adult volunteers. The hydroxyproline/creatinine ratio was found to be 15.8 +/- 4.6 mmol/mol (range 7.0-27.3) with no significant sex-related difference.     

Acyclic adenine nucleoside phosphonates in plasma determined by high-performance liquid chromatography with fluorescence detection.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and (R,S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) are selective antiviral agents with activity against a broad spectrum of DNA viruses and retroviruses. A highly sensitive method has been developed to determine concentrations of these compounds in human plasma. Plasma samples containing PMEA, HPMPA, or FPMPA were treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivatives. Chromatographic separation combined with excitation at 254 nm and fluorescence detection at 425 nm allowed quantification of PMEA, HPMPA, and FPMPA within a concentration range of 0.25-4000 mumol/L. This method proved useful in accurately determining low PMEA concentrations in sera from PMEA-treated monkeys and cats. The assay should be applicable to the quantification of PMEA, HPMPA, and FPMPA in plasma and urine of humans treated with any of these drugs.     

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.     

Quantitation of urinary hydroxypyridinium cross-links from collagen by high-performance liquid chromatography.

Pyridinoline and deoxypyridinoline are intermolecular cross-links in mature collagen in bone and cartilage. The urinary excretion of the two compounds correlates well to bone turnover. A fast, sensitive, and accurate isocratic ion-pairing reverse-phase high-performance liquid chromatography method for measurement of pyridinoline and deoxypyridinoline in urine has been established. Intra- and inter-assay precision were 5-7% and 12-14%, respectively. Recovery for pyridinoline was 97.4% and for deoxypyridinoline 94.3%. The detection limit was 0.4 pmol. Pyridinoline:creatinine and deoxypyridinoline: creatinine ratios in healthy subjects, were 38.8 nmol:mmol and 13.0 nmol:mmol, respectively. Increased values of both cross-links were observed in children, in the age group 20-29 in both sexes, and in post-menopausal women.     

变性高效液相色谱技术在β地中海贫血分型诊断中的应用

目的 探讨变性高效液相色谱技术(DHPLC)在快速诊断β地中海贫血及分型中的应用价值.方法 采用外周血红细胞平均体积(MCV)、红细胞分布宽度(RDW)、红细胞脆性和Hb电泳4项指标相结合的方法筛选地中海贫血可疑标本226份.运用PCR反向斑点杂交技术(PCR-RDB)和DHPLC对226份可疑标本进行基因分型确诊.结果 226份可疑静脉血标本中,经PCR-RDB和DHPLC确诊的β地中海贫血为69份,两种方法检测缺失和突变的基因型别完全一致,占地中海贫血筛查总人数的30.5%.其中CD41/CD42(-TCTT)移码突变37例(54%);IVS-Ⅱ-654(C→T)插入序列突变12例(17%);TATA-28(A→G)转录突变10例(15%);CD17(A→T)无义突变5例(7%);CD71/CD72(+A)移码突变5例(7%).结论 DHPLC可快速、高效和准确地对β地中海贫血进行基因分型诊断.     

超速离心-高效液相色谱测定血清高密度和低密度脂蛋白胆固醇

目的 建立一种新的预期用作参考方法的高密度脂蛋白胆固醇（HDLC）和低密度脂蛋白胆固醇（LDLC）准确测定方法。方法 血清与含2-琉基乙醇（ME）和脯氨酸的溴化钠溶液混合，使背景密度为1．063，超速离心分离高密度脂蛋白（HDL）；血清与含脯氨酸的溴化钠溶液混合，使背景密度为1.006，超速离心分离HDL和低密度脂蛋白（LDL）；用高效液相色谱测定两种脂蛋白组分胆固醇。结果 ME和脯氨酸可有效消除脂蛋白（a）对超速离心法分离HDL的影响；新方法测定HDLC和LDLC的总变异系数分别为0．96％～2、07％和0．65％～1．12％。结论 建立血清HDLC和LDLC准确测定方法，方法可靠、精密、简便，可望用作HDLC和LDLC测定参考方法。     

Simple and rapid determination of serum carbapenem concentrations by high-performance liquid chromatography

Although several high-performance liquid chromatography (HPLC) methods for the determination of serum concentrations of carbapenems have been reported, they are complicated and involve column switching. We established a simple and rapid HPLC method for the determination of serum concentrations of carbapenems, and this method is suitable for routine use in the clinical field. With our HPLC method, the serum concentrations of five commercially available carbapenems could be determined by changing only the methanol/phosphate-buffer ratio in the mobile phase. Serum levels of imipenem, panipenem, and meropenem in mice could be monitored when these carbapenems (20 mg/kg) were administered subcutaneously with cilastatin (20 mg/kg). These results suggest that our simple and rapid HPLC method for the determination of the serum concentrations of carbapenems is useful for pharmacokinetic/pharmacodynamic (PK/PD)-based determination of carbapenem dosage regimens.     

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.     

A simple, isocratic high-performance liquid chromatography assay for linezolid in human serum.

A rapid high-performance liquid chromatography (HPLC) assay for the detection of linezolid in human serum was developed. The method used a Hypersil 5ODS stationary phase. The mobile phase was 1% ortho-phosphoric acid, 30% methanol, 2 g/L heptane sulphonic acid, pH 5. UV absorbance detection was used (lambda(max) 254 nm). Samples were prepared by mixing with acetonitrile and an injection volume of 20 microL was used. The inter- and intra-day assay reproducibility were assessed. Assay linearity, specificity and accuracy were investigated. The detection limit and recovery of linezolid from serum were determined. In addition, the stability of linezolid, stored under a variety of conditions, was assessed. The retention time of linezolid was c. 6.5 min. The intra- and inter-day reproducibility was good and the assay was linear across the therapeutic range. Serum recovery was c. 100% at all concentrations tested. The detection limit was 0.1 mg/L and the assay was accurate. The assay was specific as there was no significant interference with the linezolid peak. Linezolid was demonstrated to be stable. This rapid assay is ideal for busy clinical laboratories with basic HPLC equipment.     

Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction

This rapid, reproducible method for separating and determining individual alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum is based on high-performance liquid chromatography with a weak anion-exchange column (SynChropak AX 300). The isoenzymes so resolved are detected by using an on-line enzyme reaction followed by spectrophotometric monitoring at 405 nm of the 4-nitrophenol formed. Complete diagnostic profiles of the various isoenzymes present in normal and pathological sera are obtained within 20 min. The mean (and SD) normal concentrations of the bone B1 and intestinal isoenzymes in serum of adults were 3.7 (4.3) and 4.5 (3.9) U/L, respectively (n = 14), and of the bone isoenzyme B2 and liver isoenzymes L1 and L2, 5.8 (8.6), 33.0 (10.6), and 12.0 (4.8) U/L, respectively (n = 17). Concentrations of the B2 and L1 isoenzymes in adults over age 40 years differed significantly from those in adults younger than 40 years, that of bone isoenzyme being lower (P less than 0.05) and that of the liver isoenzyme being higher (P less than 0.001) in the younger adults.     

Cefotaxime levels in ventricular cerebrospinal fluid, determined by bioassay and by high-performance liquid chromatography

Five series of cerebrospinal fluid (CSF) samples, obtained from external ventricular drains (EVD) of 4 neurosurgical patients with cefotaxime treatment were tested simultaneously by high-performance liquid chromatography (HPLC) and microbioassay using E. coli V 6311/65 as test organism. Higher cefotaxime (CTX) concentrations in CSF were measured by the microbioassay method in 4 of the 5 series, reflecting the microbioassay being influenced by increasing amounts of desacetyl-cefotaxime (DAC) during the post-application interval. Decrease of CTX levels in CSF was consistently faster in tests performed by HPLC than those using microbioassay. The clinical efficacy in gram-negative bacillary meningitis is to be explained by levels of the parent compound CTX in CSF which are several times higher than the minimal inhibitory concentrations (MICs) of most enterobacteriaceae.     

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.     

Particulate (high-molecular-mass) and soluble alkaline phosphatase in urine determined by high-performance liquid chromatography

We detected in urine by HPLC two enzyme fractions of alkaline phosphatase (AP, EC 3.1.3.1), soluble and particulate, analogous to those in serum. The second fraction was eluted with high-salt-content eluent at the same elution time as high-molecular-mass, or particulate, AP in serum. AP characterization in urine from a patient with acute rejection crisis showed a greater sensitivity of the particulate form to treatment with L-phenylalanine, which suggests a higher content of intestinal-type AP in the particulate form. The soluble fraction showed a more liver-type AP behavior. Changes in the chromatograms after the sample was treated with 1-butanol and Triton X-100 support a membrane origin of the particulate AP. Urinalyses from patients with acute renal disease showed increased activity of soluble and particulate AP, with a relatively greater increase of particulate AP.     

反相高效液相色谱法测定虫草制剂中核苷类化合物的含量

目的 建立反相高效液相色谱法(RP-HPLC)测定虫草制剂中腺苷和尿苷含量的方法 .方法 采用水浴温浸和加热回流的方法 ,制备供试品溶液.色谱柱为Hypersil C18柱(200 mm×4.6 mm)流动相为pH 6.5磷酸盐缓冲液:甲醇(85:15),检测波长为260 nm.结果 方法 的平均加样回收率:腺苷为98.9%,RSD为1.4%(n=6);尿苷为97.9%,RSD为1.5%(n=5),腺苷在23.2～928.0mg/L范围内线性关系良好,尿苷在16.0～640.0 mg/L范围内线性关系良好.结论 此方法 简便、准确,重现性好,可作为判定虫草制剂质量的一种方法 .