Liquid-chromatographic measurement of creatinine in serum and urine

We describe the adaptation of a "high-performance" liquid chromatographic method for determination of creatinine in serum and urine. The proposed method is simple, rapid, precise, and accurate. The retention time for creatinine can be varied simply by changing the KH2PO4 concentration in the mobile phase: acetonitrile/aqueous KH2PO4 (1/4 by vol). Within-day precision (CV) was 1.2-3.6% in serum chromatographed with an internal standard, and 2.3-2.8% in serum when an external standard was used. Between-day precision (CV) was 1.3-2.1% in serum and 1.3-2.7% in urine (with an external standard). Analytical recoveries of creatinine added to serum were 94-100% for the method with an internal standard, 95-103% with an external standard.     

Polarization fluoroimmunoassay of biopterin and neopterin in human urine

A polarization fluoroimmunoassay has been developed for the routine determination of biopterin and neopterin levels in human urine. The method employs fluorescein-labeled biopterin and neopterin. The assay is fast (incubation time: 2 min) and no separation step is required prior to measurement of fluorescence polarization. Linearity, recovery and precision were satisfactory. Estimations of biopterin and neopterin levels in human urine samples closely correlated with those obtained by radioimmunoassay.     

Liquid-chromatographic measurement of riboflavin in serum and urine with isoriboflavin as internal standard

We describe how riboflavin can be precisely and accurately measured in serum and urine. A structural analog, isoriboflavin, is used as an internal standard. Urine samples are prepared by adding the internal standard and trichloroacetic acid. For serum, proteins are denatured with trichloroacetic acid. A simple Sep-pak treatment of the supernate removes contaminating interferents. Within- and between-run precision is reflected by respective CVs of 2.2 and 4.9% for urine at 180 micrograms/L and 4.4 and 7.3% for serum at 10 micrograms/L. The standard curve is linear far beyond the concentrations encountered in serum and urine. The detection limit is estimated to be 10 micrograms/L and 1 microgram/L for urine and serum, respectively. The normal reference interval, as determined from 50 results for each matrix, is 36 to 349 micrograms/g of creatinine (mean 112 micrograms/g) for urine and 5.5 to 14.4 micrograms/L (mean 8.8 micrograms/L) for serum. Both distributions are skewed. The method is suited for routine use.     

Liquid-chromatographic determination of ciprofloxacin in serum and urine

We describe the liquid-chromatographic determination of ciprofloxacin in patients' serum and urine. Serum samples were prepared by precipitating protein with perchloric acid. Urine samples were diluted 100-fold with mobile phase. The mobile phase, consisting of pH 3 phosphate buffer/acetonitrile/methanol (81/5/14, by vol), was pumped through a mu Bondapak C18 reversed-phase column at 1.5 mL/min. Fluorescence of the effluent was monitored, at wavelengths for excitation and emission of 270 and 440 nm, respectively. Standard curves were linearly related to concentration from 0.08 to 10 mg/L for serum, 1 to 20 mg/L for urine. The procedure was evaluated in a clinical setting to determine its usefulness in studying the pharmacokinetics of ciprofloxacin in patients with concurrent diseases and receiving multiple drug therapies.     

血清新喋呤、生物喋呤检测及其在尿毒症患者中的应用

目的探讨尿毒症患者血清中新喋呤（NP）和生物喋呤（BP）的浓度变化及其临床意义。方法采用反相高效液相色谱-荧光检测（RP-HPLC-FD）的方法,分别测定正常对照组、尿毒症组透析前后血清中的NP、BP浓度,并进行比较。结果尿毒症组血清中的NP、BP浓度和NP／BP比值均显著高于正常组（P〈0．01）,透析后血清中的NP、BP浓度显著低于透析前血清中NP、BP浓度（P〈0．01,P〈0．05）,而NP／BP比值差异无显著性（P〉0．05）。结论在尿毒症患者中,血清中的NP、BP浓度可作为炎性介质激活的灵敏指标,同时可作为判断肾功能好坏的标志物。     

Liquid-chromatographic assay of cefmenoxime in serum and urine

This is a simple, precise liquid-chromatographic procedure for determining cefmenoxime in patients' serum and urine. p-Anisic acid is used as the internal standard. Protein is precipitated from 0.5 mL of serum or dilute urine with 100 microL of perchloric acid. The clear supernate is injected directly onto a mu-Bondapak CN reversed-phase column. The mobile phase is acetate buffer, pH 3.8 (25 degrees C). The flow rate is 2.5 mL/min. Column effluent is monitored at 254 nm. Extraction recovery from serum averaged 74.6%. Calibration curves were linear from 0.5 mg/L, the lower limit of quantification, to 100 mg/L. We present cefmenoxime concentrations in serum from a patient being treated for pneumonia. The procedure was evaluated in the clinical setting to determine its applicability to the study of cefmenoxime pharmacokinetics in critically ill patients.     

Liquid-chromatographic determination of fleroxacin in serum and urine

This highly sensitive, accurate, and reproducible HPLC method for determining fleroxacin in human serum and urine makes use of a common C18 column, a fluorescence detector, and an internal standard. Serum samples require a simple extraction procedure; urine must be diluted. The method, which we have used extensively for pharmacokinetic assessment of fleroxacin in patients, measures concentrations as low as 5 micrograms/L.     

血清新喋呤、生物喋呤检测及其在尿毒症患者中的应用

目的探讨尿毒症患者血清中新喋呤(NP)和生物喋呤(BP)的浓度变化及其临床意义。方法采用反相高效液相色谱-荧光检测(RP-HPLC-FD)的方法,分别测定正常对照组、尿毒症组透析前后血清中的NP、BP浓度,并进行比较。结果尿毒症组血清中的NP、BP浓度和NP/BP比值均显著高于正常组(P<0.01),透析后血清中的NP、BP浓度显著低于透析前血清中NP、BP浓度(P<0.01,P<0.05),而NP/BP比值差异无显著性(P>0.05)。结论在尿毒症患者中,血清中的NP、BP浓度可作为炎性介质激活的灵敏指标,同时可作为判断肾功能好坏的标志物。     

Determination of neopterin in serum and urine

Concentrations of neopterin, a product of activated macrophages, in serum from 662 apparently healthy individuals (ages 1 to 97 years, median 22 years) were measured by radioimmunoassay and the results statistically analyzed. Consistent with prior investigations on the urinary excretion of neopterin, we found no significant sex dependence, but values for subjects younger than 18 or older than 75 years were significantly higher. Renal clearance of neopterin in nine healthy individuals was 218 (SD 44) mL/min, which suggests that the kidneys have an active role in neopterin excretion. Results for neopterin concentrations measured in serum by RIA and "high-performance" liquid chromatography (HPLC) are consistent, but for urinary neopterin the concordance between methods was weak. Therefore, the RIA should be used only for measuring neopterin in serum. In comparison of the clinical utility of serum neopterin and urinary neopterin/creatinine concentrations, in patients with gynecological tumors, the latter values (measured by HPLC) discriminated slightly better between patients with favorable and unfavorable prognoses.     

Liquid-chromatographic measurement of catecholamines and metabolites in plasma and urine

In this general method for measuring the concentrations of neurotransmitters in body fluids by "high performance" liquid chromatography (HPLC), the procedures for extracting different biogenic amines from body fluids vary, but the basic chromatographic conditions are the same for all. We use citric/formic acid buffer as the mobile phase, a C18 column, and an electrochemical detector. Specificity is better than in previously reported methods. Because of its excellent cost efficiency, we consider this to be the method of choice for quantifying biogenic amines.     

HPLC法在小儿尿液新蝶呤和生物蝶呤检测中的应用

目的 建立HPLC法测定小儿高苯丙氨酸血症(HPA)患者尿液中新蝶呤(N)和生物蝶呤(B)含量的方法,以帮助临床对经典型苯丙酮尿症(PKU)和四氢生物蝶呤(BH4)缺乏症的诊断和鉴别诊断.方法 选用Cosmosil 5C18-AR-Ⅱ色谱柱(4.6 I.D.×250 mm),甲醇-水(8∶92)为流动相,用三维荧光检测器检测.结果 新蝶呤线性范围为1.60～40.0 ng/μl,生物蝶呤线性范围为1.73～43.2 ng/μl;最小检测限为0.16 ng/μl和0.1728 ng/μl.样品测定的变异系数(CV)值分别为2.18%和1.70%;原尿样品前处理时氧化的最佳时间为30 min;原尿样品不便于保存,氧化后可于室温或4℃冰箱保存3 d.结论 本方法准确、可靠、简便易行,灵敏度高,方法学实验结果满意,适用于临床HPA患儿的鉴别诊断.     

Liquid-chromatographic determination of dolichols in urine

A reversed-phase "high-performance" liquid chromatographic assay for dolichols-18, -19 and -20 in urine is described. Dolichols are extracted from urine by using C18 cartridges and are chromatographed with a mobile phase consisting of 2-propanol/methanol, the effluent being monitored at 210 nm. The useful lower limit of sensitivity for quantification is 4 pmol (5 ng) of each dolichol per 5-microL injection, corresponding to 1.6 nmol (2 ng) per liter of urine. Heneicosaprenol is satisfactory as the internal standard. Peak heights and the amounts of dolichols applied to the column are linearly related from 4 to 110 pmol. Mean analytical recovery was 71%. For three different concentrations the mean within-assay CV was 6.4%, the between-assay CV 11%. The normal reference interval of total dolichols found for healthy adults was 17-101 micrograms/24 h (n = 30) or 1.9-11 micrograms per millimole of creatinine (n = 39). I determined the distribution of the main dolichols in urine and applied the assay also for samples from alcoholics.     

Liquid-chromatographic measurement of cyclosporin A and its metabolites in blood, bile, and urine

Using solid-phase extraction columns and "high-performance" liquid-chromatographic (HPLC) analysis, we could determine cyclosporin A and nine of its metabolites in blood, bile, and urine. To facilitate calculations of concentrations of cyclosporin A and its metabolites from the chromatograms, we used cyclosporin D as internal standard. For the HPLC analysis we used two sequential 250-mm analytical columns filled with reversed-phase octyl (C8) sorbent, eluting with a concave gradient of water, adjusted to pH 3.0 with phosphoric acid, and acetonitrile. Peaks were detected at 205 nm. For characterization of the chromatographic peaks, we isolated, by semi-preparative HPLC, 32 fractions representing peaks potentially related to cyclosporin A metabolites and re-injected them into the HPLC system under the same conditions as authentic cyclosporin A metabolites. Analytical recovery was 70-80%. The inter-assay CV for bile was 7.2%, for urine 12.3%. The method was used for routine monitoring of cyclosporin A and its metabolites.     

Liquid-chromatographic determination of 4-hydroxyproline in urine

In this method for 4-hydroxyproline in urine, hydroxyproline is derivatized with 4-chloro-7-nitrobenzofurazan, with subsequent estimation by reversed-phase "high-performance" liquid chromatography. The ranges for excretion of free and total hydroxyproline while the subjects were ingesting unrestricted diets were 2-29 and 122-374 mumol/24 h (n = 21), respectively, with no significant sex-related difference. A comparison with results by colorimetry indicated no significant differences: mean (n = 18) concentrations (mumol/L) of hydroxyproline in urine were 180 (SD 149) by the present method, 163 (SD 166) by colorimetry. For protein hydrolysate the respective values were 5.9 (SD 2.7) and 6.7 (SD 2.9).     

Liquid-chromatographic determination of total hydroxyproline in urine

In this method for quantifying 4-hydroxyproline in human urine, 50 microL of urine is hydrolyzed, derivatized with phenylisothiocyanate (PITC), and then quantified by reversed-phase HPLC with ultraviolet detection. The detection limit in urine is 373 pg of hydroxyproline per 50-microL injection. The total CVs for high- and low-concentration pools are 5.3% and 3.9%, respectively (10 runs in 10 days). The standard curve of the assay is linear over a range of 0 to 22 nmol per injection. We estimated the normal range for hydroxyproline excretion in men on an unrestricted diet to be 123-308 mumol/24 h. We also report hydroxyproline concentrations in patients with metastatic bone disease and cirrhosis of the liver.     

Liquid-chromatographic determination of total hydroxyproline in urine

A reversed-phase "high-performance" liquid chromatographic assay for total hydroxyproline in urine is described. The urine samples are hydrolyzed overnight with acid, evaporated, solubilized, and derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl chloride. The derivatives are chromatographed with a solvent gradient consisting of sodium acetate buffer and acetonitrile, the effluent being monitored at 436 nm. The useful lower limit of sensitivity for quantification is 13 pmol of hydroxyproline per 5-microL injection, corresponding to 33 mumol/L of urine. Either glutamine or cysteic acid is satisfactory as the internal standard. Peak heights and the amounts of hydroxyproline applied to the column are linearly related from 13.3 to 266 pmol. Mean analytical recovery was 83%. For four different concentrations the mean within-assay CV was 9.0% and the between-assay CV 12%. The normal reference interval found for 31 healthy adults was 31 to 177 mumol/24 h per square meter of body surface. We compared results for 10 samples from patients.     

Liquid-chromatographic determination of vanillylmandelic acid in urine

Urinary vanillylmandelic acid (VMA) was determined by "high-performance" liquid chromatography with fluorometric (LC-F) and amperometric (LC-EC) detection. Urine samples were first purified on a small, open-bed, reversed-phase preparatory column. VMA and the internal standard (iso-VMA) were then separated by reversed-phase ion-pair liquid chromatography. Analytical recovery of VMA was high (98.3%, SD 3.3%, n = 8), and concentrations measured by LC-F and LC-EC were in excellent agreement (r = 0.996). The LC-F chromatograms of urine samples had fewer late peaks; however, detection limits were lower (15 vs 120 micrograms/L) for the LC-EC method. Typical concentrations of 1-10 mg/L in urine can be measured easily with either method.     

Liquid-chromatographic measurement of urinary monosaccharides

We describe "high-performance" liquid chromatography of urinary monosaccharides on a macroporous anion-exchange resin (Hitachi 3013N), with acetonitrile/water as eluent. The separated xylose, arabinose, D-fructose, mannose, galactose, and D-glucose were detected by means of a post-column reduction reaction of tetrazolium blue. Absolute limits of detection were about 10 ng of monosaccharide. Lactose can also be measured.     

Comparison of serum and urine neopterin concentrations in patients with HIV-1 infection

Urine and serum neopterin concentrations are now widely used to monitor patients with HIV-1 infection. However, there are no published studies comparing the levels in urine and serum, and relating both to the patients' immune status. Urine and serum neopterin concentrations correlated closely in our study population of 37 HIV-1 seropositive patients (34 homosexuals, 3 drug addicts) and 10 HIV-1 seronegative homosexuals. Our data further show that urine and serum neopterin concentrations correlate almost identically with the clinical and immunological presentation of HIV-1 infected individuals, as expressed by the Walter Reed Staging classification. In addition, there was no difference between the correlation of neopterin concentrations in either serum or urine with the Quetelet indices. It will depend on the clinical situation whether blood or urine sampling is preferred. Collection and handling of urine samples from HIV infected patients is less risky to health care personnel in HIV settings.     

Liquid-chromatographic determination of cephalosporins and chloramphenicol in serum

A "high-performance" liquid-chromatographic technique involving a radial compression module is used for measuring chloramphenicol and five cephalosporin antibiotics: cefotaxime, cefoxitin, cephapirin, and cefamandol. Serum proteins are precipitated with acetonitrile solution containing 4'-nitroacetanilide as the internal standard. The drugs are eluted with a mobile phase of methanol/acetate buffer (30/70 by vol), pH 5.5. Absorbance of the cephalosporins is monitored at 254 nm. Standard curves are linear to at least 100 mg/L. The absorbance of chloramphenicol is monitored at 254 nm and 280 nm, and its standard curve is linear to at least 50 mg/L. The elution times for various other drugs were also determined, to check for potential interferents.