Curvilinear-gradient high-performance liquid chromatography for identification of mycobacteria.

Over a 1-year period, 502 mycobacterial cultures submitted to the Microbial Diseases Laboratory were identified by high-performance liquid chromatography (HPLC) in parallel with standard biochemical methods. Identification by HPLC using a curvilinear gradient was achieved by comparing the chromatograms of the unknown cultures to chromatograms for known reference strains, together with calculation of peak height or peak area ratios, as necessary. The overall agreement between HPLC and biochemical identification was 97.2%. In addition, 7 of 12 cultures of Mycobacterium bovis were identified by HPLC as the BCG strain. Of 111 cultures biochemically identified as members of the M. avium complex (MAC), 108 were confirmed as MAC by DNA probe and 106 were confirmed by HPLC. Of the latter 106, 58 probe-positive strains were identified as M. avium, 38 were identified as M. intracellulare, and 10 were identified as Mycobacterium sp. strain "X" by HPLC. Of the remaining five nonchromogenic cultures, four had MAC-like chromatograms that did not match any in our library sufficiently to permit definitive identification. Of the latter four, two were confirmed as MAC strains by DNA probe and two were not. The last of the cultures biochemically identified as MAC (1 of 111) was a mixture of MAC and non-MAC strains. Overall, only 2 of 502 cultures yielded results by HPLC that differed from those obtained by standard biochemical methods. The HPLC result was confirmed in both cases by an independent national reference laboratory. In the 12 instances in which HPLC did not provide identification, the chromatograms were either uninterpretable or did not match available reference chromatograms. These findings show that the identification obtained by HPLC concurs well with that obtained by both the standard biochemical methods and the DNA probes. Thus, identification by HPLC provides mycobacteriology laboratories with a reproducible and specific method for accurate and timely identification of most medically important mycobacteria.     

Routine application of high-performance liquid chromatography for identification of mycobacteria.

Mycolic acid analysis by high-performance liquid chromatography (HPLC) was introduced in our laboratory as the routine technique for identifying all clinical isolates of mycobacteria referred to us. HPLC identified 96.1% of the 1,103 strains analyzed, whereas the biochemical procedures and/or the commercial DNA probes identified 98.3% of strains, for an overall agreement of 94.4%. Compared with the probes, there was 100% specificity and 98.9% sensitivity for Mycobacterium tuberculosis identification. HPLC allowed early detection and identification of the rare mycobacterial species M. haemophilum, M. malmoense, M. shimoidei, and M. fallax as well as uncharacteristic strains of M. simiae. After 18 months of routine use, HPLC proved to be reliable, easy to perform, rapid, and less costly than other identification methods.     

Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.     

Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures.

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.     

Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.     

Determination of moniliformin by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) was investigated as a technique for determination of moniliformin, a toxic secondary metabolite of various Fusarium species. Two HPLC procedures gave satisfactory results. In the first procedure, separation was achieved on a strong anion exchange column (10 microns, 4 mm id x 25 cm) with an eluting solvent consisting of 0.01 M sodium dihydrogen phosphate (pH 5.0) and a flow rate of 1.0 mL/min. The second procedure made use of paired ion chromatography on a reverse phase column (10 microns, 4 mm id x 25 cm). Elution was done at 2 mL/min with 0.005 M tetrabutylammonium hydrogen sulphate in a mixture containing 8% methanol and 92% 0.1 M sodium phosphate buffer (pH 7.0). Moniliformin was detected in both procedures by ultraviolet absorbance at 229 nm. The lower detection limit of pure moniliformin was 1 ng per injection. Water extraction of moniliformin from dried Fusarium cultures grown on maize was found to be efficient, giving a 95% recovery from a spiked sample. Peak height and retention time reproducibility was good for both procedures. In the analysis of maize containing on the order of 1 mg/kg moniliformin, background interference made interpretation of chromatograms difficult. The cleanup procedure giving the most promising results is described. HPLC methods have been applied successfully in screening fungal cultures for moniliformin production and for monitoring the steps in isolating moniliformin from mold material.     

Identification of carcinogenic tryptophan pyrolysis products in human bile by high-performance liquid chromatography

The carcinogenic tryptophan pyrolysis products 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) have been demonstrated to be present in human bile through use of a high-performance liquid chromatography (HPLC) method. The method consists of the acid-induced release of the carcinogens from bile components and their extraction with methylene chloride and subsequent quantification by HPLC. In seven subjects who had received catheterization of the bile duct and external biliarydrainage, the average amounts of Trp-P-1 and Trp-P-2 excreted daily in the bile were 408 fmol/day (n = 7) and 864 fmol/day (n = 7), respectively. In one subject, furthermore, significant daily changes of these carcinogen levels in bile and plasma were confirmed during 2 weeks of observation. These results indicate that one of the excretory pathways of these carcinogens is via bile. Our data also may suggest that Trp-P-1 and Trp-P-2 are derived from everyday foods.     

Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography.

The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for less than 80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of atypical mycobacteria by thin-layer chromatography of their surface antigens.

The knowledge that the surface (Schaefer) antigens of certain smooth-colony atypical mycobacteria are multiglycosylated C-mycosidic peptidoglycolipids was used to devise a sensitive thin-layer chromatographic (TLC) procedure for the identification of Mycobacterium avium/M. intracellulare/M. scrofulaceum serotypes. TLC maps of the type-specific peptidoglycolipids from 17 of the 31 serotypes are presented. The primary use of the technique is to corroborate results obtained by seroagglutination. Without the aid of seroagglutination, the TLC procedure almost invariably requires the availability of reference strains or the specific peptidoglycolipids derived therefrom.     

Allelic discrimination by denaturing high-performance liquid chromatography

Ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles allows the resolution of single-stranded DNA molecules of identical size (<100 nucleotides) that differ in a single base. Allelic discrimination is obtained by injecting short DNA amplicons containing the genetic variants of interest into an adequately preheated mobile phase that results in the instantaneous complete denaturation of the PCR products. All possible transitions and transversions other than C-->G can be typed accurately. The method complements the discovery of single-nucleotide polymorphisms by means of HPLC based heteroduplex detection under partially denaturing conditions and allows their rapid genotyping without the need of adding a reference chromosome.     

Determination of lamotrigine in human plasma by high-performance liquid chromatography.

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 microm Zorbax C8 column. Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm. The method was linear in the range of 1-20 microg/ml, with a mean coefficient of determination (r2=0.998). The limit of detection was 0.6 microg/ml and the lower limit of quantitation was 1 microg/ml using 200 microl of plasma. Within- and between-day accuracy and precision were below 6% at all analysed concentrations except at the limit of quantitation. No interfering peaks were found by commonly monitored antiepileptic drugs. Recovery was found to be > or =99%. Satisfactory performance was obtained in the evaluation of epileptic patient samples, whose results of plasma concentration measurements are briefly discussed. We conclude that this is a reliable method for the routine monitoring of lamotrigine concentration in plasma in the clinical setting.     

Purification of fatty acid methyl esters by high-performance liquid chromatography.

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane-isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Identification by high-performance liquid chromatography of immunoreactive substance P released from isolated rat spinal cord

An attempt was made to identify the immunoreactive substance P (SP) released from isolated rat spinal cords using reversed-phase high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA) for SP. Soaking the spinal cords of newborn rats in Krebs solution containing 90 mM K⁺ evoked a release of immunoreactive SP as well as of GABA and glycine in a calcium-dependent manner. Capsaicin also evoked a release of immunoreactive SP but not of GABA and glycine. The immunoreactive SP released from rat spinal cords by high K⁺ or capsaicin was analyzed by HPLC. A single peak was detected by RIA whose elution position coincided with that of synthetic SP.     

Determination of N-methyl-D-aspartate in tissues of bivalves by high-performance liquid chromatography.

The natural occurrence of N-methyl-D-aspartate (NMDA) is limited to the foot muscle of Scapharca broughtonii; it is a well known compound for its neuroexitatory activity. This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of NMDA in biological extracts. The method involves removal of neutral and basic substances by anion-exchange chromatography and removal of acidic primary amino acids by treatment with o-phthalaldehyde before derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, followed by HPLC with isocratic elution with a selected mobile phase that separates the two diastereomers formed. The identity of the detected NMDA has been confirmed by a procedure using (-)-1-(9-fluorenyl)ethyl chloroformate as a derivatizing agent. The identification has been further supported by the disappearance of the peak of the NMDA derivative by pretreatment of the sample with D-aspartate oxidase. Application of the method has shown the presence of NMDA in several tissues of S. broughtonii and Scapharca subcrenata.     

Analysis of short-chain acids from anaerobic bacteria by high-performance liquid chromatography.

A standard mixture of 25 short-chain fatty acids was resolved by high-performance liquid chromatography, using an Aminex HPX-87 column. The acids produced in culture media by anaerobic bacteria were analyzed by high-performance liquid chromatography after extraction with ether and reextraction into a small volume of 0.1 N NaOH. The presence of fumaric acid in culture extracts of Peptostreptococcus anaerobius was confirmed by gas chromatography-mass spectrometry analysis of the trapped eluent fractions from the high-performance liquid chromatography column.     

Determination of hypericin in plasma by high-performance liquid chromatography

Hypericin, a polycyclic dianthroquinone, is one of the characteristic ingredients of Hypericum perforatum extracts (St. John's wort, HP), which has antidepressant effects. Hypericin and the internal standard (I.S.), dansylamide, were extracted from plasma utilizing solid-phase extraction (SPE). Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with fluorescence end-point detection. The calibration curve was linear over the range 5-100 ng per ml of plasma. The sensitivity for hypericin was 75 pg on column. Mean inter- and intra-assay coefficients of variation (C.V.s) over the range of the standard curve were less than 10%. The absolute recovery for hypericin averaged 72.6%.     

Rapid identification of mycobacteria using gas liquid chromatography

Methyl esters of fatty acids derived from 110 strains of previously identified mycobacteria representing 17 species and a group of unidentified rapid growers, were examined by gas-liquid-chromatography (GLC). Ten species had specific GLC profiles, which enabled accurate identification; but in 2 groups of species strains shared common profiles. M. bovis, and M. xenopi usually had specific profiles but one strain of each could not be distinguished from M. tuberculosis. The group comprising M. terrae, M. fortuitum, M. chelonei, M. flavescens and rapid growers were generally not well separated by GLC; however, 6 of 12 M. terrae strains, 2 of 3 M. flavescens, and all 5 M. fortuitum strains had specific profiles. Other strains of this group had only common peaks and by GLC were indistinguishable from each other. Using a table of specific and characteristic peaks, 34 of 54 (63%) recent isolates were correctly identified, and 18 (33%) were correctly allocated to groups sharing similar GLC profiles: only 2 isolates were wrongly identified. At present, GLC analysis provides easy and rapid identification of a majority of mycobacteria but cannot replace fully biochemical tests in the identification of medical mycobacteria.     

Determination of famotidine in human plasma and urine by high-performance liquid chromatography.

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C18 reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 microg/ml, respectively; intra- and inter-day coefficients of variation were < or =10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.     

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography.

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen -1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO2), were isolated from the basified biological matrix using a liquid-liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate-hexane-triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.