A modification of the pisano method for vanilmandelic acid using high pressure liquid chromatography

A method for the measurement of urinary vanilmandelic acid (VMA) is described based on a modification of the colorimetric procedure of Pisano. VMA is oxidized to vanillin with subsequent measurement of the vanillin by high pressure liquid chromatography with electrochemical detection (LCEC). The technique has been successfully applied to a number of human and animal urine samples. Urinary VMA values obtained by the LCEC method are in good agreement with those determined by the method of Pisano. The new method is both more selective and more sensitive than the colorimetric procedure.     

Hypoxanthine production by ischemic heart demonstrated by high pressure liquid chromatography of blood purine nucleosides and oxypurines

An isocratic high pressure liquid Chromatographic system was developed for the estimation of purine nucleosides and oxypurines in blood. Use was made of a reversed-phase column. Nucleotides derived from erythrocytes affected the separation; these compounds were removed with Al₂O₃. The recovery of the whole clean-up procedure exceeded 75%, and the lower detection limit of the assay for blood metabolites was 0.1 μmol/l. In 6 healthy volunteers, non-resting, the following blood concentrations (mean values ± S.D. in μmol/l) were observed: adenosine (< 0.1), inosine (0.2 ± 0.1), hypoxanthine (2.2 ± 1.3) and xanthine (0.2 ±0.1). In plasma and serum the total amount of these compounds was 1.9 and 5.4 times higher, respectively, presumably due to nucleotide breakdown during blood processing. The myocardial arterial-venous differences of blood purine nucleosides, oxypurines and lactate were subsequently measured in blood samples from 13 patients with angiographically documented ischemic heart disease, undergoing an atrial pacing stress test. No significant release of adenosine, inosine and xanthine by the heart was detectable in this study. The myocardial arterial-venous difference of lactate changed from 0.01 ± 0.03 mmol/1 (mean ± SEM) at rest, to ?0.10 ± 0.04 mmol/1 during pacing (p < 0.002). Relatively larger changes were observed for hypoxanthine: pacing increased the arterial-venous difference from ?0.01 ± 0.05 to ?0.51 ± 0.17 μmol/1 (p <0.02). We conclude that the high pressure liquid chromatographic assay of blood hypoxanthine is a useful tool in the diagnosis of ischemic heart disease.     

The rapid determination of erythrocyte porphyrins using reversed-phase high performance liquid chromatography

The extraction, separation and quantification of free acid erythrocyte porphyrins are described. The porphyrins are extracted from whole blood using 3:1 ethyl acetate/acetic acid (v/v) and separated using the reversed-phase mode of high performance liquid chromatography (RPLC). The compounds are detected on-line spectrofluorometrically using an excitation wavelength of 404 nm and an emission cut-off filter of 550 nm. Excellent resolution of the porphyrin free acids is achieved in < 6 min. The analytical recoveries for protoporphyrin IX and zinc protoporphyrin IX were > 90% with relative standard deviations for day-to-day analysis under 6%.     

Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry.

A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.     

Hypoxanthine-guanine phosphoribosyl transferase: assay using high performance liquid chromatography

A method is described for the estimation of hypoxanthine-guanine phosphoribosyltransferase using high performance liquid chromatography.The inosine monophosphate (IMP) generated from hypoxanthine is determined, after separation on a C₁₈ reversed phase silica column with a buffer-methanol gradient, by the absorbance at 254 nm. Simultaneous reciprocal measurement of hypoxanthine consumption is made. The assay is suitable for screening red cell lysates for hypoxanthine-guanine phosphoribosyltransferase deficiency; the results being expressed as nmol inosine monophosphate · h^?1 · mg^?1 haemoglobin.The normal range found was 94 ± 15 nmol IMP-h^?1 · mg^?1 haemoglobin and hypoxanthine-guanine phosphoribosyltransferase activities down to 1% of normal can be assayed accurately.     

Analysis of lidocaine and its dealkylated metabolites by high-pressure liquid chromatography.

A high-pressure liquid chromatographic method is presented for determining lidocaine and its dealkylated metabolites, monoethylglycinexylidide and glycinexylidide, in plasma. This method uses 20-microliter samples injected directly through a pre-column onto a C18 analytical column. It is fast, sensitive and linear over a concentration range of 1--10 microgram/ml. Since the method requires no preliminary treatment of the plasma, it provides a practical means of monitoring lidocaine and its metabolites in therapeutic situations.     

The estimation of plasma valproate by gas-liquid chromatography.

A gas chromatographic method for the plasma assay of the anticonvulsant, sodium valproate, is described. Derivatization is not necessary. 200 microliter plasma are required for a single estimation. The method involves a chloroform extraction of valproate and the internal standard, cyclopentane carboxylic acid, from acidified plasma. Gas-liquid chromatography using the stationary phase 10% SP-216-PS gives complete separation of valproate and the internal standard in eight minutes. The limit of detection is 20 mumol valproate/1 plasma (equivalent to 40 pmol on column). This is well below the lower therapeutic plasma level. The between-run precision of the method indicates a variation for each sample within+/-3% of its mean value.     

Analysis of total urinary catecholamines by liquid chromatography: methodology, routine experience and clinical interpretations of results

A simple routine method is described for simultaneous assay of total urinary adrenaline, noradrenaline and dopamine. The catecholamines are pre-purified on a small ion-exchange column, separated by reversed phase ion-pair liquid chromatography, and are quantitated by electrochemical detection. The method was routinely applied to 422 urines. Elevated values were found in four urine specimens obtained from patients with histologically proven phaeochromocytomas. Virtually no interference by endogenous or exogenous compounds was found. Values for urinary catecholamines determined by fluorimetric analysis agreed with those obtained by high pressure liquid chromatography with electrochemical detection. Within-day CVs for the compounds ranged from 5.2-11.9%, between-day CVs from 3.3-6.6%. The normal range (95% confidence level) was 20-230 micrograms/24 h for noradrenaline and 1-35 micrograms/24 h for adrenaline.     

The effect of humidity on the determination of lecithin/sphingomyelin ratio and phosphatidylglycerol by thin-layer chromatography

Cortisol in concentrated sulfuric acid forms two major fluorescing compounds which are separated by chromatography on a reversed-phase column. Based on this reaction, urine free cortisol, after double extraction was assayed by high-performance liquid chromatography with fluorescent detection. The method is sensitive, rapid, and free from interferences. The values by this method are 30% lower than radioimmunoassay. In addition to Cushing's syndrome, patients with acute pancreatitis had elevated levels of urinary free cortisol which paralleled urinary amylase levels while patients with myocardial infarction had normal levels. Urinary free cortisol showed diurnal variation with elevated values early in the morning.     

The determination of total cholesterol in serum by gas-liquid chromatography compared with two other methods

A gas-liquid chromatographic method is described for the measurement of total cholesterol in serum. The method has been found to be simple, specific and precise. The results have been compared with the results of the enzymatic method of Röschlau, P., Bernt, E. and Gruber, W. (1974) (Z. Klin. Chem. Klin. Biochem. 12, 403) and with the results of an Auto-Analyzer method based on the manual method of Huang, T.C., Chen, C.P., Wefler, V. and Raftery, A. (1961) (Anal. Chem. 33, 1405) and Ness, A.T., Pastewka, J.V. and Peacock, A.C. (1964) (Clin. Chim. Acta 10, 229). The results of the gas-liquid chromatographic and of the enzymatic method show a high degree of correlation. The results of the Auto-Analyzer method are about 0.75 mmol/l higher than those of the other two methods. The conclusion is drawn that the gas-liquid chromatographic method should be given consideration as a reference method for the measurement of total cholesterol in serum. It is a viable alternative for the generally accepted colorimetric reference method of Abell, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952) (J. Biol. Chem. 195, 357).     

Determination of glycosylated adult and foetal haemoglobins by affinity chromatography

Estimation of adult glycosylated haemoglobin by affinity chromatography was found to be quick and less dependent on ionic strength, pH and temperature than ion-exchange chromatography. Results obtained by both procedures correlated strongly (r = 0.96) but the range for normal subjects was smaller with the affinity assay. The affinity method correlated equally well with the colorimetric assay (r = 0.95). However, the method did not measure all the glycosylated forms, and only half of the glycosylated species isolated by ion-exchange chromatography was bound to the affinity resin. It also showed that the amount of glycosylated haemoglobin in cord blood is less than in adult blood.     

Endogenous levels of free and conjugated urinary 3-methoxy-4-hydroxyphenylethyleneglycol in control subjects and patients with pheochromocytoma determined by reversed-phase liquid chromatography with electrochemical detection

A sensitive and specific reversed-phase liquid-chromatography (HPLC) method for the determination of urinary free and conjugated 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) was developed. Sample preparation is minimal and the method is ideally suited for routine clinical assays of this catecholamine metabolite.Reported in this paper are excretion profiles of control subjects and patients with pheochromocytoma. Quantitative results were expressed as μg MHPG per mg creatinine, in order to eliminate variations associated with the 24-h urine collection. The levels of free and conjugated MHPG, determined in 20 control subjects ranged between 0.04–0.11 μg/mg of creatinine and 0.48–1.25 μg/mg of creatinine, respectively. The corresponding levels of free and conjugated MHPG in 8 patients with pheochromocytoma were 0.29–1.21μg/mg of creatinine and 2.47–15.98 μg/mg of creatinine, respectively.The described liquid-chromatographic analysis, coupled with the highly sensitive electrochemical detection, provides a simple and reliable method for metabolic profiling of catecholamine by-products at their endogenous levels. This offers an attractive possibility for the detection of neural crest lesions which may cause problems in diagnosis because of their small size, infrequent occurrence and symptoms similar to those of essential hypertension.     

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.     

Specific quantitation of plasma medroxyprogesterone acetate by gas chromatography/mass spectrometry

Investigation of various derivatives (O-methyloxime, trifluoroenol acetate and t-butyldimethylsilyl enol ether) coupled with the efficient preparation of a trideuterated analogue of medroxyprogesterone acetate, has allowed the development of a rapid and accurate assay for its quantitation in plasma by isotope dilution gas chromatography/mass spectrometry. High oral doses (2.8 g weekly) of medroxyprogesterone acetate are shown to lead consistently to plasma levels of less than 16 ng.ml-1.     

Quantitative analysis of serum lipoproteins by micro-scale thin-layer chromatography.

A method has been devised for the complete chemical analysis of serum lipoproteins, in which the constituents are separated by thin-layer chromatography and then measured by means of a flame ionisation detector. Since the response of the detector differs for each constituent, it is necessary to use a previously prepared calibration curve for each one. A complete analysis can be obtained from a single run on about 20 microgram of lipoprotein. However, from 5--10 chromatograms are needed for an adequate degree of precision. The method, which could be adapted to the measurement of tissue lipids, takes less than 2 h to complete. This speed and simplicity seem to give the method considerable potential for the investigation of patients with disorders of lipid transport.     

Determination of 3-mercaptolactate, mercaptoacetate and N-acetylcysteine in urine by gas chromatography

A method is described for the determination of 3-mercaptolactate, mercaptoacetate (thioglycolate) and N-acetylcysteine in urine. As these compounds are mainly excreted as their mixed disulfides with cysteine, they are first reduced to the free thiols by an insoluble polymer containing thiol groups. After purification by chromatography on an organomercurial adsorbent, the compounds are converted to benzyl derivatives by extractive alkylation and determined by gas chromatography. The identity of the compounds analyzed was verified by mass spectrometry. It was demonstrated that mercaptolactate and mercaptoacetate are almost entirely excreted as their mixed disulfides with cysteine, whereas appreciable amounts of N-acetylcyteine are present as the symmetrical disulfide and the free thiol. The urinary excretion of the compounds from healthy human beings was also studied.     

Determination of phosphatidylglycerol in amniotic fluid by a simple one-dimensional thin-layer chromatography method

Phosphatidylglycerol (PG) in amniotic fluid is the second important component of lung surfactant phospholipids and may be clinically useful in assessing fetal lung maturity in utero. Although methods for PG determination are available, there are shortcomings in clinical application. We developed an alternative reliable onedimensional thin-layer chromatography (TLC) method for separating and quantitating PG in amniotic fluid. A mini-TLC plate (8 × 10 cm) was prepared from silica gel H containing 5% ammonium sulfate. The plate was first developed in tetrahydrofuron/dimethoxymethane/methanol/2N ammonium hydroxide (30.0:20.6:5.6:3.0, v/v) and then in chloroform/methanol (60:9, v/v) in the same direction. PG was clearly separated from other phospholipids and neutral lipids, even when large amounts of other phospholipids were present on the TLC plate. The density of the charred PG was directly proportional to the amount of PG up to 8 nmol. The content of PG in nmol in the specimen can be quantitated by comparing with a standard PG. Up to 10% of blood serum or 3% meconium showed no detectable PG, nor did these substances affect PG quantitation in amniotic fluid. This method is sensitive and accurate. It is also time-saving and economical.     

Determination of plasma 3-methoxy-4-hydroxyphenyl-glycol by pulsed electron capture gas chromatography.

An improved method is described for determining picogram quantities of 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma of humans and of other species. The method makes use of gas-liquid chromatography and electron capture detection. Low level nonlinearity of detector response was corrected by operating the detector in the pulsed rather then the customary steady state mode. Detector overloading was prevented by heat coagulation of plasma proteins and subsequent ultrafiltration. Sensitivity was significantly enhanced by utilizing a derivatizing agent carrying a higher number of electrophores. Baseline conditions are described and control values for plasma MHPG of human volunteers, Rhesus monkeys and rats are presented.