Biomedical Chromatography

Llambias, Elena B. C.; Luo, Jinli

doi: 10.1002/(SICI)1099-0801(199607)10:4<155::AID-BMC577>3.0.CO;2-Zpmid: 8831958

Methods for the analysis of phenformin and its metabolite by high‐performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high‐performance liquid chromatographyPelectrospray ionization mass spectrometry (HPLCPESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLCPESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4‐hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and Km was 4 μM

Fisher, Daniel H.; Broudy, Marc I.; Fisher, L. Megan

doi: 10.1002/(SICI)1099-0801(199607)10:4<161::AID-BMC579>3.0.CO;2-3pmid: 8831959

A method was developed for quantitating 9‐carboxy‐11‐nor‐Δ9‐tetrahydrocannabinol in human urine as part of the process for validating an automated enzyme immunoassay for marijuana metabolites. Sample cleanup was accomplished using a mixed‐mode solid‐phase extraction. 9‐Carboxy‐11‐nor‐Δ9‐tetrahydrocannabinol and the internal standard, brominated 9‐carboxy‐11‐nor‐Δ9‐tetrahydrocannabinol, were quantified using high‐performance liquid chromatography with electrochemical detection (+0.85 V). The linear range for this method is 0.012–0.20 μg/mL. No interference was seen for 22 drugs and metabolites. The pooled relative standard deviation is 4.1% (n=27) for the quality control samples. This method was compared to gas chromatography with mass spectrometry by linear regression analysis. The slope of the line is 1.00±0.05 (standard error), the intercept is approximately zero, the coefficient of determination is 0.994, and the standard error of the estimate is 0.006 μg/mL.

Khedr, Alaa

doi: 10.1002/(SICI)1099-0801(199607)10:4<167::AID-BMC584>3.0.CO;2-#pmid: 8831960

Parameters affecting the separation of amino acids on different RP‐HPLC columns were studied. Six amino acids were separated on Zorbax TMS, Zorbax CN, Zorbax ODS and Zorbax C8, using 1.8×10−3 M copper sulphate at pH 4.1 as aqueous mobile phase. The best separation was shown by Zorbax TMS followed by Zorbax CN. The column performance was maintained by serial washing after 6 h continuous work with aqueous 10−4 M disodium edetate, 10−4 N sulphuric acid, water and gradient elution with aqueous methanol. The separation mechanism was interpreted. The method was applied for separation and quantification of aztreonam and L‐arginine in Azactam vials.

Delbeke, F. T.

doi: 10.1002/(SICI)1099-0801(199607)10:4<172::AID-BMC588>3.0.CO;2-1pmid: 8831961

Urinary concentrations of the β‐antagonist oxprenolol and some of its major human metabolites were determined following oral administration of a dose of 160 mg to five fasted horses. Quantitation was performed by gas chromatography–mass spectrometry (GC–MS) in the selected ion mode (SIM) by monitoring ion m/z 466 of the heptafluorobutyric derivatives. As early as 2 h after dosage oxprenolol could be detected in hydrolysed urine and remained detectable up to 24 h. Maximum urinary concentrations and excretion rates were obtained between 2 and 12 h. After 12 h only 2.8% of the administered dose was excreted as conjugates of oxprenolol and major human metabolites including 4‐OH‐oxprenolol and 5‐OH‐oxprenolol. These metabolites were detectable up to 48 h.

Cirimele, Vincent; Kintz, Pascal; Mangin, Patrice

doi: 10.1002/(SICI)1099-0801(199607)10:4<179::AID-BMC586>3.0.CO;2-Npmid: 8831962

We have tested several solvents for the decontamination of control hair contaminated by cocaine. Methylene chloride was the most efficient solvent, eliminating more than 60% of contaminating cocaine. Two washes were used, additional washes were not effective in removing more cocaine. Sixteen hair and three pubic hair samples obtained from drug abusers were tested by different extraction procedures: acid, alkaline and enzymatic hydrolyses and extraction by methanol. This study revealed that acid and enzymatic hydrolyses gave higher extraction recoveries for drugs than methanolic extraction. After alkaline hydrolysis and methanolic extraction, extracted samples were dirty and the chromatograms obtained showed an important interfering background. We concluded that acid hydrolysis is the extraction method of choice.

Santa, Tomofumi; Kimoto, Kousuke; Fukushima, Takeshi; Homma, Hiroshi; Imai, Kazuhiro

doi: 10.1002/(SICI)1099-0801(199607)10:4<183::AID-BMC581>3.0.CO;2-Ipmid: 8831963

A new fluorescent reagent for carboxylic acids, 4‐(N‐hydrazinoformylmethyl‐N‐methyl)amino‐7‐N,N‐dimethylaminosulphonyl‐2,1,3‐benzoxadiazole (DBD‐CO‐Hz) was synthesized and its applicability as a precolumn derivatization reagent in high‐performance liquid chromatography (HPLC) was examined. DBD‐CO‐Hz reacted with saturated fatty acids in the presence of a condensing agent, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and pyridine at room temperature for 30 min to give fluorescent products. The reaction solution was subjected to a reversed‐phase HPLC and detected fluorometrically at a wavelength of 550 nm with an excitation of 440 nm. The detection limits of the derivatives were at low fmol range on column.

Conaway, Concetta A.; Fried, Bernard; Sherma, Joseph

doi: 10.1002/(SICI)1099-0801(199607)10:4<186::AID-BMC582>3.0.CO;2-6pmid: 8831964

High‐performance thin‐layer chromatography (HPTLC) was used to determine neutral lipids and free fatty acids in the digestive gland–gonad complex (DGG) and faeces of Biomphalaria glabrata. Three populations of B. glabrata were given varying food intakes for one week. They were either unrestricted (lettuce ad libitum plus a weekly feeding of Tetramin), restricted (15–17 mg of lettuce every two days), or starved (no food). Samples were prepared by extraction in chloroform:methanol (2:1), followed by a Folch wash (0.88% aqueous KCl). The major lipids detected in the DGG were triacylglycerols, free fatty acids and free sterols. Free fatty acids and free sterols were the major lipids detected in the snail faeces. Analysis by TLC–densitometry showed a statistically significant difference in the DGG levels of triacylglycerols and free sterols between the unrestricted and restricted groups, as well as between unrestricted and starved groups. Analysis of faecal samples revealed a significant difference in free fatty acids between unrestricted and starved groups on the seventh day.

Kimoto, Kohsuke; Gohda, Ryoya; Murayama, Katsuhisa; Santa, Tomofumi; Fukushima, Takeshi; Homma, Hiroshi; Imai, Kazuhiro

doi: 10.1002/(SICI)1099-0801(199607)10:4<189::AID-BMC585>3.0.CO;2-Ppmid: 8831965

The highly sensitive detection of four near‐infrared (near‐IR) fluorescent dyes, methylene blue, pyridine 1, oxazine 1 and 3,3′‐diethylthiadicarbocyanine iodide (DTDCI) by high‐performance liquid chromatography (HPLC) with post‐column peroxyoxalate chemiluminescence (PO‐CL) detection utilizing bis(4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide was examined. The detection limits for methylene blue, pyridine 1, oxazine 1 and DTDCI were 120, 27, 31, 0.19 fmol on‐column at a signal‐to‐noise ratio of 2, respectively. The sensitivity for DTDCI was 250 times that obtained by HPLC with the conventional fluorescent detection.