Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol: acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.     

High-pressure liquid chromatography of alpha-keto acid 2,4-dinitrophenylhydrazones

Various α-keto acids were separated as their 2,4-dinitrophenylhydrazine derivatives by ion-pair, reverse-phase, high-pressure liquid chromatography. Excellent baseline resolution was obtained for a seven-component homologous series of α-keto acid dinitrophenylhydrazones at increasing carbon-chain length. Branched-chain keto acids were also separated. Resolution of syn and anti isomers of the α-keto acid derivatives was possible. Pyruvate from biological material was located and identity confirmed by an enzymic peak shift technique. Monitoring at 366 nm permits low-level (nanogram) amounts of keto acids to be detected. Ion pair versus ion exchange is discussed with regard to the mechanism of chromatographic separation.     

Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography-mass spectrometry

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is approximately 20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.     

Quantitative micro determination and isolation of plasmalogen aldehydes as 2,4-dinitrophenylhydrazones

A micro spectrophotometric procedure for the quantitative determination of plasmalogen aldehydes is described which utilizes simultaneous methanolysis and formation of 2,4-dinitrophenylhydrazones. After isolation of the hydrazones by thin-layer chromatography, the aldehydes can be regenerated, reduced, acetylated, and then analyzed by gas-liquid chromatography. Identification of the plasmalogen aldehydes obtained from rumen holotrich protozoa is described.     

Rapid thin layer chromatographic separation of para-phenylazophenyl thiohydantoins of amino acids

3 (p-phenylazophenyl) 2-thiohydantoins of amino acids were identified on silica gel plates in 30 mins using ascending thin layer chromatographic technique. Recovery percentages of these amino acid derivatives were calculated and observed that the method of preparation needed modification.     

Analysis of positional isomers of monounsaturated fatty acids by high performance liquid chromatography of 2,4-dinitrophenylhydrazones of reduced ozonides

A method is described for quantifying the positional isomers in monounsaturated fatty acid methyl ester (FAME) fractions. The procedure involves the preparation of 2,4-dinitrophenylhydrazones (DNPH) of the fragments generated during reductive ozonolysis of FAME, class isolation of the aldehyde and aldehyde ester DNPH, and separation of the aldehyde ester derivatives by high performance liquid chromatography (HPLC). The high extinction coefficient of the DNPH provides for a sensitive assay which is linear for a large range of components over a concentration range of 0.075-5 nmol/component, and the stability of the DNPH permits the independent analysis of the aldehyde and aldehyde ester fragments generated during reductive ozonolysis. The reductive ozonolysis-DNPH-HPLC method developed is as sensitive, reproducible, and accurate as reductive ozonolysis-gas-liquid chromatography and does not suffer from some of the drawbacks of the classical procedure.     

The quantitation of rat serum esterases by densitometry of acrylamide gels stained for enzyme activity.

Normal rat sera were electrophoresed on polyacrylamide gels. The gels were then stained for (a) esterase activity and (b) protein content. Subsequent analysis on a densitometer permitted quantitative estimation of both enzyme activity and protein concentration. (The specific activity (per mg of protein) of individual serum esterases were then calculated.) Two previously unreported esterases (Bands A and D), were identified and partially characterized. When carefully prepared, non-activated plasma was used, no differences in enzyme activity were found compared to serum.     

Adenosine tetraphosphate analysis: Polyethyleneimine (PEI) thin layer purification and firefly-extract liquid scintillation quantitation

The role and sources of adenosine tetraphosphate (AP₄) have not been extensively identified because its separation and quantitation necessitates analyses on a microscale. This article illustrates a method of separation and quantitation previously not known for this purpose. The use of polyethyleneimine (PEI) thin layer chromatography with an unusual spotting media (separation time = 25–50 min), combined with noncoincident liquid scintillation counting via luciferin-luciferase light emission in a kinetically delayed manner to produce a startling increase (16–20 fold) in emitted light illustrates the uniqueness of the system. We are able to separate and detect picomole quantities of adenosine tetraphosphate. The use of this system should enable researchers to identify the importance of adenosine tetraphosphate.     

Improved quantitation of plasma lipids by direct gas-liquid chromatography

A preliminary digestion of total plasma lipid extracts with phospholipase C, which converts the lysolecithins, lecithins, and sphingomyelins into monoglycerides, diglycerides, and ceramides, respectively, has been shown to facilitate subsequent determination of the plasma lipids by gas-liquid chromatography. A further improvement in the chromatographic elution pattern results from acetylation or trimethylsilylation of the liberated alcohol moieties prior to injection into the chromatograph. If tridecanoin is used as internal standard, quantitative estimates can be rapidly obtained for plasma lysolecithins, free cholesterol, lecithins, sphingomyelins, cholesteryl esters, and triglycerides, as well as for free fatty acids. Other plasma lipids do not occur in sufficiently high concentrations to interfere with the analysis. The determination requires 0.1-0.5 ml of plasma and about 6 hr of processing, but many samples can be processed at a time.     

Rapid analysis and quantitation of PCR products by high-performance liquid chromatography

HPLC utilizing a high efficiency anion-exchange column provides a rapid and easily automated technique for the qualitative and quantitative analysis of subnanogram to microgram amounts of DNA fragments generated by the PCR. The accuracy, precision and linearity of this method exceed those attainable with electrophoretic techniques. In addition, because of the nondestructive nature of HPLC, the desired PCR amplification product can be purified for subsequent utilization. Thus, liquid chromatography extends the utility of the PCR technique to those applications requiring precise quantitation.     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.     

Rapid detection and quantitation of Clostridium perfringens enterostoxin by counterimmunoelectrophoresis.

Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 microgram of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific and easy for the detection and quantitation of enterotoxin.     

Rapid detection and quantitation of Clostridium perfringens enterostoxin by counterimmunoelectrophoresis.

Conditions for detection and quantitation of Clostridium perfringens enterotoxin by counterimmunoelectrophoresis are described. As little as 0.2 microgram of enterotoxin per ml could be detected. The test was found to be rapid, sensitive, specific and easy for the detection and quantitation of enterotoxin.     

Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance

A specific strain of Chlorella protothecoides has been studied in heterotrophic fermentation for increasing cell growth rate and lipid content for biodiesel production. For optimizing the process of fermentation to reduce costs of alga-based biodiesel production, rapid determination of lipid content in microalgal cells is critical. Nile Red (NR) staining and time-domain nuclear magnetic resonance (TD-NMR) have been investigated to quantitate the lipid content in C. protothecoides. Both methods were found feasible and simpler than gravimetric methods that are commonly employed. The TD-NMR method showed better agreement (R² = 0.9973) with the measured values from lipid extraction experiments than the NR staining method (R² = 0.9067). Additionally, the smaller standard deviations of the samples (≤ 0.36) analyzed by TD-NMR revealed that the method is accurate and reproducible. The application of TD-NMR for lipid quantitation in C. protothecoides opens up the possibility of determining lipid content in algal fermentation precisely and quickly.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of colchicine in human plasma

A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050-10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.     

Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of metformin in human plasma using phenformin as internal standard has been developed and validated. Sample preparation of plasma involved acidification with acetic acid, deproteination with acetonitrile and washing with dichloromethane. Samples were then analyzed by HPLC on a short Nucleosil C18 column (5 microm, 50 mm x 4.6 mm i.d.) using a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate pH 7.0 (20:20:60, v/v/v) delivered at 0.65 ml/min. Detection was performed using an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring (MRM) mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The assay was linear over the range 1-2000 ng/ml with intra- and inter-day precision of <8.6% and accuracy in the range 91-110%. The limit of detection was 250 pg/ml in plasma. The method was successfully applied to a clinical pharmacokinetic study of an extended-release tablet of metformin hydrochloride (500 mg) administered as a single oral dose.