Artifacts in chromatography: an overview.

A brief overview of the field of analytical artifacts is provided, with examples of solvent impurities, stabilizers, polymer additives, and problems relating to Teflon, glassware, and laboratory contaminants.

Solid-phase extraction of chloramphenicol with graphitized carbon black.

The sorption and desorption properties of graphitized carbon black (GCB) were evaluated for enrichment of highly polarizable chloramphenicol (CP) from biological fluids. The distribution of CP between GCB and water lay strongly toward GCB. Among the organic solvents examined, methanol gave the lowest adsorption coefficient. The optimum packing amount of GCB, and the optimum volume and flow-rate of the eluent were deduced from the breakthrough curve and methanol elution curve, respectively. The inner diameter of GCB extraction columns was chosen from the sorption efficiency curve obtained by plotting CP recovery against column inner diameter. The solid-phase extraction method using GCB was applied to the assay of CP in serum samples in the concentration range of 5 to 50 micrograms/ml.

Gas chromatographic-mass spectrometric analysis of volatile constituents in saliva.

Present methods for the development of metabolic profiles are limited to the use of headspace techniques and solvent extraction methods. A new method for the development of saliva profiles which provides information complementary to existing analyses has been developed. The results of the developed methodology provide a reliable, reproducible method for metabolic profiling. Gas chromatographic-mass spectrometric analysis of the volatile constituents provided positive identification of 39 compounds. Application of the developed protocol toward the investigation of saliva as a vehicle for the non-invasive detection of certain pathological states, specifically diabetes mellitus and liver disorders, may be possible.

Determination of acrylamide in nerve tissue homogenates by electron-capture gas chromatography.

Acrylamide in biological samples can be determined by gas chromatography with electron-capture detection after conversion to its 2,3-dibromopropionamide derivative. The derivatization is carried out in aqueous solution, plasma or tissue homogenates by ionic bromination and the reaction conditions and sample clean-up are described. The detection limit corresponds to 9.5.10(-12)g of acrylamide on column or 8.4.10(-9)g in the final biological extract (0.5 ml). The overall recovery of acrylamide spiked samples at the nanogram level exceeds 80%. It was found that the accumulation of free acrylamide in the sciatic nerve distal region of rats intoxicated with acrylamide was less than 2-8 ppm.

The role of organic volatile profiles in clinical diagnosis.

The organic volatile constituents of biological fluids contain clinically useful diagnostic information for the recognition of metabolic disorders in man. To gain access to this information, it was necessary to develop the methodology for reproducibly stripping the trace concentrations of volatiles from biological fluids (dynamic headspace, gas phase-stripping, solvent extraction, and the transevaporator technique), to separate the complex extracts by high-resolution capillary column gas chromatography, and to develop computer-aided data-handling and pattern-recognition techniques for analyzing the immense amount of information generated. The normal and pathological organic volatiles identified by gas chromatography--mass spectrometry in urine, serum, and breast milk are tabulated. Clinical applications of the above techniques to the study and diagnosis of diabetes mellitus, respiratory virus infection, renal insufficiency, and cancer are described.

The determination of bifunctional compounds. IX. A selective reaction for the determination of guaifenesin in plasma by gas chromatography.

Guaifenesin after extraction from plasma with an organic solvent can be selectively derivatized with 2,4-dichlorobenzeneboronic acid and determined by gas chromatography with electron-capture detection. The detection limit for guaifenesin was 15 ng ml-1 for a 2.0-ml plasma sample. The mass spectra of the boronate derivatives of guaifenesin and mephenesin, used as internal standard, show good molecular ions with characteristic modes of fragmentation useful for their identification.

The determination of steroids with and without natural electrophores by gas chromatography and electron-capture detection.

The response of the electron-capture detector to organic compounds is poorly defined, and the steroids are no exception to this observation. For those steroids which are naturally electron-capturing, the structures of the electrophores will be defined. Other steroids can be made electron-capturing by the formation of appropriate derivatives. Some new or infrequently used reagents for this purpose (flophemesyl ethers, t-bulflophemesyl ethers, pentafluorophenylhydrazone derivatives and halogen-substituted aromatic boronic acids) are described.

Volatile metabolites in sera of normal and diabetic patients.

The profiles of volatile metabolites in serum samples from normal individuals and from individuals with diabetes mellitus with varying degrees of polyneuropathy have been studied. The transevaporator procedure was used to obtain sample extracts which were chromatographed on a highly efficient glass column coated with Silar 10C (106 m x 0.25 mm I.D.). Differences in profiles between normal subjects and diabetic subjects on no drug therapy were noticed. However, correlations between the severity of the neuropathy and the concentrations of certain ketones could not be established. Compounds present both in diabetic and normal sera have been identified by mass spectrometry.

Capillary column gas chromatographic profile analysis of volatile compounds in sera of normal and virus-infected patients.

Using a transevaporator sampling technique, the volatile profiles from 70 microliter of serum were obtained by capillary-column gas chromatography. The complex chromatograms were interpreted by a combination of manual and computer techniques and a two-peak ratio method devised for the classification of normal and virus-infected sera. Using the K-nearest neighbor approach 85.7% of the unknown samples were classified correctly. Some preliminary results indicate the possible use of the method for the assessment of virus susceptibility.

Simultaneous determination of antiarrhythmia drugs by high-performance thin-layer chromatography.

A method is described for the determination of antiarrhythmia drugs in serum by high-performance thin-layer chromatography. Baseline separations are achieved for all the drugs and clozapine, an internal standard, in two developments with solvents of different polarity. Lidocaine and diphenylhydantoin are scanned at 220 nm after the first development. Procainamide, propranolol and quinidine are scanned at 290 nm after the second development. The relative standard deviation of the determination varies between 3 and 14% depending on the nature of the drug and its concentration.

The determination of sucrose in molasses by high-performance thin-layer chromatography.

A method is described for the determination of sucrose in molasses by high-performance thin-layer chromatography. A baseline separation of sucrose is achieved in about 40 min using acetone--water (90:10) as developing solvent. The relative standard deviation of determinations by this method is about 2.2%. Results agree with those obtained by isotope dilution analysis of the same molasses samples.

High-performance thin-layer chromatographic determination of psychopharmacologic agents in blood serum.

High-performance thin-layer chromatography affords a rapid, sensitive method for determination of psychopharmacologic agents in blood serum samples. Quantitation of the representative drugs chlorpromazine, amitriptyline, nortriptyline, imipramine, and desipramine at levels as low as 5 ng/ml is demonstrated by scanning the developed thin-layer plates with a chromatographic spectrophotometer in the ultraviolet absorption mode. Neither derivatization prior to, nor color development after chromatographic separation is required to achieve sensitivity and reproducibility of determinations.

Nickel gas chromatographic columns: an alternative to glass for biological samples.

Nickel tubing may be substituted for glass in the fabrication of gas chromatographic columns for use with samples of biological interest. Comparisons of separations of mixtures of steroids, narcotic alkaloids, phenothiazines, and amphetamines on stainless stell, glass, and nickel packed columns showed little or no observable sample decomposition on glass or nickel as contrasted to complete loss of certain compounds on stainless steel. The nickel columns are easily prepared, durable, economical, and not subject to breakage.

Identification of dihydrothiazoles in urine of male mice.

The compounds 2-isopropyl-4,5-dihydrothiazole and 2-sec-butyl-4,5-dihydrothiazole were identified in urine of male mice. Their excretion in mouse urine is sex-dependent. In the urine of female mice the two dihydrothiazoles are either absent or present only in trace amounts. The analytical procedure includes the adsorption of the volatile mouse urine constituents on Tenax GC, their gas chromatographic separation and the detection of the sulfur compounds by the sulfur-specific detector. The identified compounds were synthesized. Natural and synthetic compounds had the same mass spectrometric properties and retention data.

Column elution and concentration of volatile compounds in biological fluids.

A new solvent elution method is described for the isolation and concentration of volatile metabolites in biological fluids. The procedure involves elution of the sample with a solvent through a micro-column of an adsorbent into glass-wool and evaporation of the excess solvent in a stream of helium at room temperature. The volatiles are revocered by subsequent heat desorption into a chromatogrpaphic system. Both elution and evaporation are made in a single step using a novel sampling device. Since the technique is based on liquid-solid adsorption chromatography, the adsorbent-solvent system which gives the maximum yield of volatile constituents was studied. The method is simple, highly efficient and reproducible, requiring only small volumes of biological fluids (less than 100 mul).

Profiles of volatile metabolities in body fluids.

A method for the analysis of volatile metabolites present in plasma, urine, breast milk and amniotic fluid collected from mother-infant pairs has been developed which requires only 100 mul of plasma, 3 ml of urine, 20 mul of breast milk and 500 mul of amniotic fluid. After extraction with diethyl ether, the volatile compounds were absorbed on glass wool in a special concentration tube and subsequently desorbed and transferred to a 100-m nickel capillary column for analysis by gas chromatography and gas chromatography-mass spectrometry. The separations, carried out by temperature programming, were complete in 90 min.

Methionine dipeptide stationary phases for the resolution of enantiomers.

N-Trifluoroacetyl-L-methionyl-L-methionine cyclohexyl ester as well as the disulfide and disulfone derivatives of this phase have been synthesized. The methionine phase exhibits improved properties as a stationary phase for the separation of amino acid enantiomers as compared to other dipeptide stationary phases and can be used at temperatures ranging from 70 to 150 degrees. The sulfoxide phase, however, was incapable of resolving the enantiomeric derivatives. Physical properties of the sulfone compound limit its use as an effective resolving agent.