A new high-performance liquid chromatographic procedure to quantitate hemoglobin A1c and other minor hemoglobins in blood of normal, diabetic, and alcoholic individuals

A new HPLC procedure is described for the separation and quantitation of minor human Hb variants. The cation exchanger, Synchropak CM 300, is a silica support with a bonded polymeric coating of carboxylic acid residues. The chromatogram is developed with 0.03M Bis-Tris-KCN buffers, pH 6.40, and a Na-acetate gradient increasing from 0 to 0.1125M. As many as 11 minor Hbs (including Hb A2) can be isolated. Some of these have been identified through rechromatography of the minor Hb zones obtained by Bio-Rex-70 chromatography. Quantitation of Hb A1c and Hb A2 is readily accomplished. Hb F0 and an unidentified minor Hb often observed in red cells of alcoholics co-chromatograph with Hb A1c. The method has been applied to blood samples of 10 normal adults, 13 diabetic patients, and nine alcoholic subjects. An excellent correlation exists between the Hb A1c percentages and the levels of Hb A1 determined by microcolumn chromatography. Some other minor Hbs, identified as components 9 and 10, which are (at least in part) Hb A0 with glucose attached to the alpha chains, are present in increased amounts in the blood of diabetic patients and others may be observed in patients who subject themselves to (severe) alcohol abuse. It is suggested that the new procedure is well-suited for detailed studies of the minor Hbs in patients with various abnormalities in carbohydrate metabolism.     

A high-performance liquid chromatographic method for determination of hypoxanthine in cord plasma

A reversed-phase high-performance liquid chromatographic method was developed for the analysis of hypoxanthine in cord plasma. Cord plasma was precipitated with ammonium sulphate and after centrifugation the supernatant was injected into the chromatographic system. Separation of hypoxanthine was optimal with phosphate buffer (20 mmol/l, pH 7.65-7.75) as the mobile phase. Limit of determination using 200 microliter of the sample was found to be 1 mumol/l with a coefficient of variation of less than 10%. The imprecision of this method at the 10 mumol/l level was about 2%. The method described in this report for determination of hypoxanthine in cord plasma would also be suitable for determination of hypoxanthine in other biological fluids in clinical investigations. We have also determined reference values for hypoxanthine in citrate-preserved cord plasma. Rigorous sampling conditions and sample handling are important to achieve accurate results.     

谷胱甘肽的快速荧光检测法

目的建立一种灵敏、特异、稳定、快速的谷胱甘肽（ＧＳＨ）检测方法。方法用二硫苏糖醇还原,邻苯二醛柱前衍生,用长（１５０ｍｍ ）或短（３０ ｍｍ）反相Ｃ１８柱色谱分离,再用荧光检测器测定血浆ＧＳＨ。结果用 Ｃ１８反相长柱和Ｃ１８反相短柱进行ＧＳＨ色谱分析结果基本一致,两组标准曲线的相关系数为 ０．９９８４。用短柱分析的时间为长柱的 １／７,灵敏度为＜ ２μｇ／Ｌ,在 ２～２００ｍｇ／Ｌ范围内,日内变异系数和日间变异系数均小于 １０％。正常人血浆ＧＳＨ水平为 １１．１６±４．２２ ｍｇ／Ｌ。结论用 Ｃ１８反相短柱进行ＧＳＨ色谱分析,用荧光分光光度计定量是一种灵敏、特异、快速并易于临床开展的检测方法,可用于血浆或器官组织ＧＳＨ水平的研究。     

Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples

A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.     

Microchromatography of hemoglobins. IV. An improved procedure for the detection of hemoglobins S and C at birth.

Substitution of CM-cellulose for CM-Sephadex had yielded a superior microchromatographic method for distinguishing the AS, AC, SS, SC, and CC conditions at birth. On the translucent columns of CM-Sephadex, the hemoglobin zones are somewhat diffuse. However, the compact, well-defined zones on the CM-cellulose column facilitate the interpretation of the results even though the amount of sample is only 20 per cent as great. The CM-cellulose method is as simple and rapid as the original CM-Sephadex procedure.     

High performance liquid chromatographic procedure for the simultaneous determination of theophylline, caffeine, and phenobarbital in neonates

A sensitive HPLC method is reported for the simultaneous determination of theophylline, caffeine, and phenobarbital in 100 microliters of plasma. After a single extraction of the drugs with chloroform/isopropanol (90 + 10 by volume) at low pH in the presence of an excess of ammonium sulphate they are resolved and quantified using a reversed-phase column (Spherisorb 5 ODS). The drugs are eluted with a binary-solvent gradient system (acetonitrile/phosphate buffer pH 4.6) at room temperature and monitored at 204 nm. Quantitation is based on peak-height ratio of analyte to interval standard (8-chlorotheophylline). Complete chromatographic resolution of all drugs is achieved within 15 min. The method is linear to 40 mg/l of theophylline and caffeine, and to 80 mg/l of phenobarbital. Numerous drugs and xanthine metabolites tested do not interfere.     

Assessment of the automated colorimetric and the high-performance liquid chromatographic methods for nicotine intake by urine samples of smokers' smoking low- and medium-yield cigarettes

We have compared the high-performance liquid chromatographic method with the direct barbituric acid test in an assessment of nicotine exposure. The effect of the endogenous colour in urine on the direct barbituric acid method was also studied. The applicability of methods was evaluated with urine samples from 15 smokers who smoked 5, 10 and 20 low- and medium-nicotine cigarettes per day. Assessments of nicotine intake with the methods were well correlated, although the high-performance liquid chromatographic method detects only cotinine and the direct barbituric method most of the nicotine metabolites. Before endogenous colour subtraction in the DBA method the correlation coefficient was 0.558 and after that 0.784. Both methods indicated similar changes in nicotine exposure with the change of brand and also a dose dependent relationship to the number of cigarettes smoked. The coefficients of variation for both of the methods were 3.4%. The endogenous colour determination lessened the capacity of direct barbituric acid method to one half being about 150 samples per day. With the high-performance liquid chromatographic method the capacity was about 50 samples per working day.     

Rapid large scale screening of blood donor plasma for IgA deficiency,confirmation and quantitation

Plasma from each of 20,068 blood donor samples collected for routine ABO and Rhesus grouping were screened for IgA deficiency using a rapid latex agglutination inhibition technique. 83 donors gave positive reactions with the latex screening reagent used. After elimination of the various false positives using further latex tests, 24 donors were eventually classified as true IgA deficients with an IgA level of less than 0.05 g/L, giving an incidence of 1:836.     

Column-switching high-performance liquid chromatographic method for determination of a new antiviral agent, cyclaradine, in serum.

Cyclaradine is a novel carbocyclic nucleoside with good activity against the viruses of the herpes group. To facilitate pharmacokinetic studies on cyclaradine, an automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the determination of cyclaradine in serum. Deproteinized serum containing cyclaradine was directed to an on-line extraction column which retained cyclaradine while many potentially interfering components were eluted to waste. Fourteen minutes later, the switching valve was automatically rotated, permitting an appropriate mobile phase to elute cyclaradine from the extraction column onto an analytical C18 column for further separation and UV detection. The method showed excellent linearity (r = 0.9995) for cyclaradine concentrations ranging from 0.05 to 5 micrograms/ml. It also provided good sensitivity (0.05 micrograms/ml). The assay was precise, with within-run and between-run coefficients of variation of less than 1.90%. The accuracy, expressed as differences between observed values and theoretical values, ranged from -4.12 to 4.80%. The assay involves a simple deproteinization of serum followed by a fully automated sample cleanup, eliminating long and tedious manual extraction prior to HPLC. The column-switching HPLC method has been successfully used for the determination of cyclaradine serum levels in squirrel monkeys following a single oral dose of 20 mg/kg.     

Quantification of hemoglobins S, C, and F by a magnetic affinity immunoassay

This magnetic affinity immunoassay (MAIA) quantifies hemoglobins (Hb) S, C, and F in hemolysates from adults or newborns. Monospecific antisera to the hemoglobins are covalently conjugated to magnetic beads and reacted with the corresponding 125I-labeled hemoglobin. After centrifugation to separate the free and antibody-bound 125I-labeled hemoglobin, the amount of radioactive hemoglobin in the pellet is measured. To determine the concentration of the Hb under study, the percent inhibition of the reaction is quantified. The standard curve is established by adding known quantities of unlabeled hemoglobin before adding 125I-labeled hemoglobin. The amount of Hb S, Hb C, or Hb F present in hemolysates is determined by measuring the percentage of inhibition and extrapolating the concentration from the standard curve. Results agree well with values for Hb S and Hb C obtained by "high-performance" liquid chromatography and RIA and for Hb F as measured by alkali denaturation and RIA. This assay can be completed in 1 h and is more sensitive than enzyme immunoassay or RIA: we can detect proportions of Hb S and Hb C as low as 1% of total Hb, and Hb F as low as 0.05%.     

Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma

A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C(18) column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 microg/ml for HPLC and from 0.25 to 20 microg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 microg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.     

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

Background and Objective: All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high‐performance liquid chromatography (HPLC). RhD‐negative women who deliver RhD‐positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). Materials and Methods: The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF‐positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti‐D FC was performed to quantitate fetal D‐positive cells in D‐negative women and compared with FMH estimated by AE and HbF FC. Results: AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. Conclusions: To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti‐D FC are discrepant or when there is maternal and fetal RhD compatibility.     

Rapid high-pressure liquid chromatographic method for analysis of phenoxymethylpenicillin in human serum.

A selective high-pressure liquid chromatographic method for the determination of phenoxymethylpenicillin in human serum is described. The technique is based on the single extraction of the drug from acidified serum with diethyl ether. Chloramphenicol is used as internal standard. The chromatographic system consists of a reversed-phase C-18 column; the mobile phase is acetonitrile-0.01 M potassium acetate buffer (20:80 [vol/vol]; pH 6.5). The method can accurately measure serum penicillin concentrations down to 30 micrograms/liter with 500 microliter of sample. The coefficient of variation for intraassay variability of penicillin is between 1.5 and 4.9% in the range of 0.125 to 16.00 mg/liter. The extraction efficiency is 78.5 +/- 6.8% (+/- standard deviation; n = 9), and the calibration graph is linear in the concentration range studied. Pharmacokinetic data, obtained with the present method from seven healthy volunteers, are presented.     

Anion-exchange high performance liquid chromatography method for the quantitation of nucleotides in human blood cells

An anion-exchange high performance liquid chromatography (HPLC) method is described for the quantitation of intracellular purine and pyrimidine nucleotides. With an ammonium phosphate salt and pH gradient, complete separation is achieved of all major nucleotides and several interfering substances, such as dehydroascorbic acid and NAD. For optimal resolution of the monophosphates, strict control of the equilibration pH is essential. To prevent interference by a degradation product of NADPH with the determination of GDP, the pH of the high-ionic strength buffer has to be in the range of 4.9-5.0. The use of radially compressed, prepacked cartridges filled with Partisil-10 SAX appeared to be a fast and cheap alternative for expensive stainless-steel columns. The use of ammonium phosphate buffers, in combination with precolumns filled with pellicular silica and SAX resin, and interim EDTA washes prevents baseline shift. This allows analysis at 0.01 Absorbance Units Full Scale during the entire column lifetime (about 180 analyses), which is sufficiently sensitive for the quantitation of low levels of nucleotides, especially when the amount of sample is limited.The usefulness of the presented Chromatographie system is demonstrated by the quantitation of the nucleotides in extracts of lymphocytes and neutrophils from the blood of healthy human donors. With this method nucleotide concentrations were measured, with a within-assay variation of 5–10% and an inter-donor variation of 10%.