A non-enzymic procedure for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)ethylene glycol (MHPG sulphate) in human urine using stable isotope dilution and gas chromatography—mass spectrometry

A method is described for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)-ethylene glycol (MHPG sulphate) in human urine, based on selected ion monitoring gas chromatography—mass spectrometry and using a specifically deuterium-labelled analogue of MHPG sulphate as internal standard. The procedure involves extraction of the urine sample on Amberlite XAD-2, followed by isolation of MHPG sulphate by column chromatography on Sephadex LH-20. Cleavage of the sulphate conjugate and formation of the MHPG tris(trifluoroscetate) derivative are carried out in a one-step reaction, without recourse to enzymic hydrolysis.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

VI. The concurrent estimation of the major monoamine metabolites in human and non-human primate brain by HPLC with fluorescence and electrochemical detection

A simple and sensitive method for the concurrent determination of the monoamine metabolites MHPG, DOPAC, HVA and 5HIAA in brain samples is described. After solvent extraction at acid pH, the metabolites are separated by HPLC on a C₁₈ reversed phase column using phosphate buffers. Detection and quantification are achieved using fluorescence and electrochemical detection in series. The method is applied to control samples of divers areas of human and non-human primate brain, and the distribution of results agrees well with those obtained by existing methods. The concentrations found also agreed well with literature values, and, for 5HIAA and DOPAC, with results obtained on parallel samples analysed by fluorimetry and by GC. Results for HVA however are higher than those obtained by GC, but agree well with literature values obtained by fluorimetry and by GCMS.     

Rapid non-enzymatic HPLC determination of total MHPG in human plasma

We have previously reported a method for the determination of total 3-methoxy-4-hydroxy phenylethylene glycol (MHPG) in brain, based on a simple acid-catalyzed hydrolysis. Now we extend this procedure to the determination of plasma total MHPG. The method involves the deproteinization of plasma with perchloric acid, followed by 3 minutes of an acid-catalyzed step. The hydrolysates are injected into the HPLC system, using a formic acid/methanol eluent with fluorimetric detection. Sample detection limit is below 1 ng MHPG/mL of plasma. This procedure has been used for the determination of plasma total MHPG from 109 healthy individuals of both sexes. Mean value was: 5.4 + 2.3 ng total MHPG/mL of plasma (means +/- S.D., N = 109). No sex differences were observed, and a slight correlation with age (r = 0.24, p less than 0.02) has been found. Plasma-free MHPG was also determined in a subgroup of 15 randomly chosen individuals (3.0 +/- 1.2 ng free MHPG/mL plasma, means +/- S.D.). A significant correlation was obtained with plasma total MHPG (r = 0.77, p less than 0.001, N = 15). The main advantage of the present method lays in its simplicity, since no enzymatic hydrolysis or extraction procedures are needed, being its reliability fully proven through 109 plasma total MHPG determinations.     

Isotope dilution ammonia chemical ionization mass fragmentographic analysis of urinary 3-O-methylated catecholamine metabolites: Rapid sample clean-up by derivatization and extraction of lyophilized samples

We developed a method for simultaneous quantification of the urinary 3-O-methylated catecholamine metabolites 3-methoxytyramine, normetanephrine and metanephrine by stable isotope-dilution ammonia chemical ionization mass fragmentography. Prepurification of lyophilized samples was done by simultaneous deconjugation and pentafluoropropionylation, followed by extraction and rederivatization. Compared with our previously described method, based on acid hydrolysis, alkaline extraction, derivatization and electron-impact mass fragmentography, the present method was found to be less laborious, more sensitive and presumably more accurate. New urinary excretion values were established for apparently healthy adults. The present prepurification method may prove applicable for profiling of a variety of naturally occurring mono-, di- and polyamines in biological samples.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

A rapid extraction method for detecting aflatoxin producing isolates

A rapid extraction method for screening aflatoxin producing potential ofAspergillus flavus group isolates is described. The method is performed using a moist wheat medium with ca. five infected grains extracted with 2 mL of chloroform, and using thin layer chromatography. This method was proved with 95A. flavus isolates from animal feeds.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

A fast and versatile method for extraction and quantitation of long-chain acyl-CoA esters from tissue: content of individual long-chain acyl-CoA esters in various tissues from fed rat.

A method for the extraction of acyl-CoA esters from tissue, and their subsequent analysis by HPLC is described. The lipids are removed by a two-phase extraction in a chloroform/methanol/water system. The long-chain acyl-CoA esters are extracted using methanol and a high salt concentration (2 M ammonium acetate). Reextraction of the dry residue after evaporation of extraction solvent results in low overall recoveries (20%). By adding 1 mg/ml acyl-CoA-binding protein to the extraction solvent the overall recovery was increased to 55%. The method is easy and fast to perform and is thereby suitable for analysis of a large number of samples. The advantages of the method over previously published methods are discussed.     

A method for extraction and analysis of high quality genomic DNA from ixodid ticks

Currently, no published methods describe the extraction of high molecular weight genomic DNA from ixodid ticks (Acari: Ixodidae) and commonly used methods of extraction are not well adapted for use with members of this family. A method for extraction of minimally degraded genomic DNA from ixodid ticks that can be completed in one or two days is described. The method produces DNA which is of sufficient size (>24 kb) for use in Southern analysis and which is readily digestible by restriction endonucleases. Southern analysis using a cytochrome P450 gene probe, demonstrates the success of our method with genomic DNA extracted from two species of Ixodidae, the lone star tick, Amblyomma americanum (Linnaeus) and the cattle fever tick, Boophilus microplus (Canestrini).     

Detection of 3-methoxy-4-hydroxyphenylglycol in rabbit skeletal muscle microdialysate

A high-performance liquid chromatography with electrochemical detection (HPLC-ED) method is described for determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in microdialysate from the skeletal muscle interstitial space. Using a microdialysis technique, we sampled 30 microl dialysate from the skeletal muscle interstitial space and injected dialysate directly into HPLC-ED system. The control MHPG concentration of dialysate was 213+/-18 pg/ml. The MHPG concentrations were reduced by entacapone (catechol-O-methyltransferase inhibitor, COMT), augmented by local infusion of dihydroxyphenylglycol. This system offers a new possibility for simple, rapid monitoring of MHPG as an index of COMT activity in skeletal muscle.     

Quantitation of total MHPG in the rat brain using a non enzymatic hydrolysis procedure. Effects of drugs

An acid-catalyzed procedure has been used to hydrolyze MHPG-sulfate in homogenates of rat brain. The samples (in 0.4 mol/L perchloric acid) are treated for 3 min. at 100 degrees C in a water bath and aliquots are injected into a reversed phase HPLC system. Detection is achieved fluorimetrically. The absolute detection limit for MHPG is 150 pg, which allows the reliable determination of either free or total MHPG in rat brain in concentrations down to 15 ng/g, using the described procedure. The concentration of total MHPG found in the brains of saline-treated rats are 101 +/- 21 ng/g (mean +/- S.D.) which is in a good accordance with the concentration value for the same samples obtained using a GC-MS method (115 +/- 19 ng/g). Rats treated with clonidine (300 micrograms/Kg, i.p.) or yohimbine (10 mg/Kg, i.p.) showed brain concentrations of total MHPG of 68 +/- 22 ng/g and 299 +/- 85 ng/g, respectively. The utility of this method for the analysis of brain regions or brain nuclei (e.g. locus coeruleus) is also shown.     

A semiautomated electron capture gas chromatographic assay of 3-methoxy-4-hydroxyphenylethyleneglycol in urine

A semiautomated method for the assay of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine has been developed. The method incorporates a new efficient (95%) extraction procedure combined with an automated gas chromatographic system. This system (consisting of a pulsed electron capture detector and an automatic sample injector which valves the sample between two columns) will accept continuous, unattended, sequential sample input. The limit of sensitivity of the trifluoroacetic anhydride derivative of pure MHPG is 3 pg. Analysis of samples from the same 24-h urine (n = 21) over a 2-month period resulted in a mean of 794 ng/ml with a coefficient of variation of 3.5%. MHPG levels from 12 control males and 9 females with no history of psychiatric disorder, were found to contain 1605 and 1034 μg/24 h, respectively.     

Lipid Extraction Methods for Lipomyces starkeyi

Various lipid extraction methods were applied to Lipomyces starkeyi, freeze-dried, heat-dried and intact cells. It was found that the freeze-dried cells were usually more extractable than other types of cells. A high lipid recovery was obtained by a lytic enzyme (Corticium centrifugum) treatment, conc. HCI treatment and Pedersen’s method using chloroform-methanol (1: 1). The first two methods, however, hydrolyzed phospholipids and released free fatty acids during the extraction of lipids. From the results we have obtained, the best method for lipid extraction from L. starkeyi is that in which the freeze-dried cells are extracted by the Pedersen’s method. The results obtained from the application of these methods to a hydrocarbon-grown yeast are also described.     

Methods for DNA extraction from Candida albicans

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.     

Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.     

The direct application of the polymerase chain reaction to DNA extracted from foods

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.     

Isolation and quantitative determination of some cardioactive glycosides from Digitalis lanata by high-performance liquid chromatography

A rapid extraction method followed by high-performance liquid chromatographic assay was developed for the quantitative determination of the cardioactive glycosides of Digitalis lanata. The leaf samples were extracted with water or aqueous alcohols. The simple extraction method gives a better yield than the methods described previously. Lanatoside C and its metabolites have been separated on a reversed-phase column with various mixtures of acetonitrile, methanol, and water as mobile phases for isocratic elution. Extraction and quantitative determination of lanatoside C and digoxin from a leaf sample require not more than 30 min.     

An effective, rapid and simple method for total RNA extraction from bacteria and yeast

In this work, we describe a rapid and simple method for total RNA extraction from bacteria and yeast. The method allows for the acquirement of high RNA yields while avoiding the use of phenol or other toxic reagents and is less expensive than other methods previously described. The extracted RNA is suitable for applications such as RT-PCR, Northern blot hybridization and low molecular weight RNA (LMW RNA) electrophoresis.     

Determination of urinary 4-hydroxy-3-methoxyphenylethylene glycol in man by high performance liquid chromatography with electrochemical detection

A reverse phase high performance liquid chromatographic method for determining levels of 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) in human urine that is virtually free of all interfering peaks has been developed. After addition of a homologous internal standard, enzymatic hydrolysis is performed. Samples are then placed onto columns containing AG1-X8, and the MHPG is collected in a phosphate buffer wash of the column. After ethyl acetate extraction and evaporation of the organic solvent, the dry residue is redissolved in mobile phase, and injected onto a reverse phase column. Results obtained with this assay were almost identical (101±5.6%, mean±SD, n=6) with those obtained using a gas chromatography - mass spectrometry (GCMS) assay.