Biomedical Chromatography

doi: 10.1002/bmc.1130070415pmid: N/A

doi: 10.1002/bmc.1130070401pmid: N/A

Benchaoui, H. A.; McKellar, Q. A.

doi: 10.1002/bmc.1130070402pmid: 8219694

A high performance liquid chromatographic method has been developed for the determination of rafoxanide and closantel in ovine plasma. Acetonitrile and chloroform were used for the extraction. The mean recoveries were 78.69% and 80.59% for rafoxanide and closantel, respectively. This method was applied to the characterization of rafoxanide plasma kinetics following oral administration of therapeutic doses to sheep.

Kataoka, Hiroyuki; Nakai, Kiyohiko; Katagiri, Yoshimi; Makita, Masami

doi: 10.1002/bmc.1130070403pmid: 7693088

A selective and sensitive method has been developed for the analysis of free and bound forms of O‐phosphoamino acids, such as O‐phosphoserine, O‐phosphothreonine and O‐phosphotyrosine, in urine samples by gas chromatography (GC). For free O‐phosphoamino acid analysis, the urine sample was extracted with trichloroacetic acid and run through an ion exchange column. For total (free plus bound) O‐phosphoamino acid analysis, the urine sample was hydrolysed in acid and base in order to release the O‐phosphoamino acid from peptides or proteins. O‐Phosphoamino acids in these prepared samples were converted into their N‐isobutoxycarbonyl trimethyl ester derivatives and then measured by GC with flame photometric detection (FPD‐GC). The calibration curve was linear in the range of 10–500 ng for each O‐phosphoamino acid, and the detection limits were 30–80 pg as injection amounts. O‐Phosphoamino acids in the urine samples could be selectively determined by the FPD‐GC method without any influence from coexisting substances. The recoveries of O‐phosphoamino acids added to urines and urine hydrolysates were 90–98% and relative standard deviations were 1.5–8.0%. By using this method, we investigated an age dependence of O‐phosphoamino acid excretion in normal subjects.

Smolenski, Ryszard T.; Yacoub, Magdi H.

doi: 10.1002/bmc.1130070404pmid: 8219695

A reversed phase high performance liquid chromatographic technique is presented allowing purine catabolite determination in a whole blood extract without a prior purification step. The method was applied to determine the timing and the profile of myocardial nucleotide catabolite release during reperfusion of the transplanted human heart. Samples of arterial and coronary sinus blood collected at various times within 1 h after aortic declamping during heart or heart‐lung transplantations were used for nucleotide catabolite determination. Massive release of inosine and hypoxanthine from the heart was demonstrated. Production of adenosine was also shown but there was no liberation of xanthine or uric acid. Nucleotide catabolite release was greatest in the first 5 min (coronary sinus‐arterial difference = 15–20 μM), but was still significant after 30 min of reperfusion. The determination of inosine and hypoxanthine–‐major catabolites released–‐was found to be reproducible in coronary sinus blood (coefficient of variation < 10%). However, immediate protein precipitation afer blood sample collection was necessary, as rapid metabolism of both exogenous and endogenous adenosine and inosine was demonstrated.

Song‐gang, Ji; Qing‐hong, Kong; Xiu‐lu, Li; Ping, Li

doi: 10.1002/bmc.1130070405pmid: 8219696

A gas chromatographic method for the determination of mexiletine in human plasma is described. Mexiletine was simultaneously extracted and derivatized with carbon disulphide for separation and quantitation on a glass column (1.5 m × 3 mm i.d.) packed with 1.5% OV‐1 coated on 80–100 mesh Shimalite W (201D). The method required only 0.5 mL of plasma and could detect as little as 10 ng of mexiletine. It has been applied to the study of the pharmacokinetics of mexiletine in healthy volunteers.

Massias, L.; Postaire, E.; Regnault, C.; Hazebroucq, G.

doi: 10.1002/bmc.1130070406pmid: 8219697

Formation of free radicals and lipoperoxidation occur at the onset of cellular damage. These effects are produced during normal metabolism and in pathological states. The peroxidation of polyunsaturated fatty acids, i.e. linoleic acid and linolenic acid, which are both cellular membrane compounds, induces ethane and pentane formation in pulmonary air exhalation. These two volatile hydrocarbons can be considered as potential lipoperoxidation markers. Methodological difficulties limit the use of these gases for assessment of free oxygen radical activity but we have developed and validated a non‐invasive technique. A study was performed with ten healthy volunteers.

Charles, Bruce; Chulavatnatol, Suvatna

doi: 10.1002/bmc.1130070407pmid: 8219698

A simple high performance liquid chromatographic method with ultraviolet detection at 229 nm is described for quantitation of amoxycillin in plasma. After deproteination of plasma samples with perchloric acid and adjustment of the pH to 4.9, the supernatant was injected onto a reversed phase C18 column, using acetonitrile: phosphate buffer (0.01 M, pH 7.4) (1:25 v/v) as the mobile phase. Amoxycillin and the internal standard, cefadroxil, were eluted at 23 min and 12 min, respectively, without interference from endogenous substances. Processed samples were stable for at least 24 h at room temperature which permitted automated batch processing overnight. Calibration plots of the amoxycillin to cefadroxil peak‐height ratio vs. amoxycillin concentration were linear (P < 0.0001; r⩾0.995) from 0.25 mg/L to at least 16.0 mg/L. Between‐day and within‐day imprecision (CV) ranged between 3.7% and 17.7%. Absolute recovery for amoxycillin and cefadroxil exceeded 82%. The application was demonstrated by the analysis of amoxycillin in human plasma after a single orall dose of amoxycillin (250 mg) suspension.

Toyo'oka, Toshimasa; Chokshi, Hitesh P.; Givens, Richard S.; Carlson, Robert G.; Lunte, Susan M.; Kuwana, Theodore

doi: 10.1002/bmc.1130070408pmid: 8219699

Fluorescence and chemiluminescence analyses of amino acids and thiols derivatized with 2‐fluoro‐4,5‐diphenyloxazole (DIFOX) and 2‐chloro‐4,5‐bis(p‐N,N‐dimethylaminosulphonylphenyl)oxazole (SAOX‐CI) were investigated. Thirteen diphenyloxazole (DIOX)‐derivatized amino acids were separated within 38 min by a linear gradient elution from 100% A (0.05 M phosphate (pH 7.0):CH3CN (75:25)) to 100% B (0.05 M phosphate (pH 7.0):CH3CN (1:1)) over 30 min and an isocratic elution of 100% B for 30 min. The detection limits (S/N = 2) with fluorescence detection were in the range of 19–64 fmol. Thiols derivatized with SAOX‐CI were separated by an isocratic elution using 0.1 M H3PO4:CH3CN (65:35) and detected fluorimetrically. The detection limits (S/N = 2) of reduced glutathione, N‐acetylcysteine, 2‐mercaptopropionylglycine, cysteine, homocysteine and captopril were 1.2, 1.5, 1.9, 5.7, 6.4 and 7.9 fmol, respectively. Peroxyoxalate chemiluminescence (CL) intensities of sulphonyl‐5‐N,N‐dimethylaminonaphthalene (DNS), SAOX and DIOX derivatives were compared using three different oxalate esters (DFPO, TCPO and TDPO) by flow injection analysis. The relative chemiluminescence intensity (RCL) of SAOX‐proline and DIOX‐proline were 76–80% and 19–25% of DNS‐proline (100%), respectively. Other SAOX and DIOX derivatives showed lower CL intensities ( < 12%). Extremely low CL intensities were obtained for the fluorescent tagging reagents (<0.11%) and their hydrolysis products (<0.80%). Secondary amino acids and peptides, derivatized with DIFOX in aqueous media at room temperature for 1 h, were detected using DFPO/H2O2. TCPO/H2O2 and TDPO/H2O2 after separation by high performance liquid chromatography. The detection limits (S/N = 2) of hydroxy‐L‐proline, L‐proline, L‐prolyl‐glycyl‐glycine and L‐prolyl‐L‐leucine with the three oxalate esters were 10–17 fmol, 22–34 fmol, 50–61 fmol and 80–123 fmol, respectively.

Nakashima, Kenichiro; Kawaguchi, Shinki; Akiyama, Shuzo; Schulman, Stephen G.

doi: 10.1002/bmc.1130070409pmid: 8219700

A high performance liquid chromatographic method with chemiluminescence detection for the determination of penicillin G and ampicillin is reported. The method is based on the enhancement of the luminol chemiluminescence with β‐lactam antibiotics. The linear relationship was obtained between the peak height and the concentration of penicillin G or ampicillin up to 15 nmol per 20 μL injection. Detection limits were 1 nmol for penicillin G and 0.5 nmol for ampicillin with a signal‐to‐noise ratio of 2. Relative standard deviations for five replicate measurements of 4 nmol/injection each of penicillin G and ampicillin were 2.1 and 2.3%, respectively.