Quantification of methamphetamine, amphetamine and enantiomers by semi‐micro column HPLC with fluorescence detection; applications on abusers' single hair analysesAl‐Dirbashi, Osama Y.; Kuroda, Naotaka; Wada, Mitsuhiro; Takahashi, Masakatsu; Nakashima, Kenichiro
doi: 10.1002/1099-0801(200008)14:5<293::AID-BMC2>3.0.CO;2-Kpmid: 10960827
Achiral and chiral semi‐micro column high‐performance liquid chromatographic methods with fluorescence detection to determine methamphetamine and amphetamine in human hair are described. These compounds were extracted into 5% trifluoroacetic acid (TFA) in methanol, derivatized with 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)‐benzoyl chloride and separated either on a 250 × 1.5 mm i.d. octadecyl‐silane (ODS) or a 150 × 2 mm i.d. OD‐RH column. Linear calibration curves extending over a wide range of concentration that covers the practical samples were obtained for amphetamine, methamphetamine and their enantiomers (r = 0.999). Resolution values for amphetamine and methamphetamine enantiomers were 3.4 and 1.1, respectively. Intra‐ and inter‐day variations of both the methods were not larger than 8.9% expressed as relative standard deviations (n ≥ 5). The limits of detection at a signal‐to‐noise ratio of 3 obtained by both the methods were in the range of 1.0–4.7 fmol/5 µL injection with the achiral method being more sensitive. Abusers' hair samples were analyzed by the two methods and only the S(+)‐enantiomers were found in eight Japanese abusers' hair samples. The achiral method was used to study the concentrations of these compounds in single black and white hair strands of abusers. Copyright © 2000 John Wiley & Sons, Ltd.
Capillary electrophoretic analysis of brimonidine in aqueous humor of the eye and blood sera and relation of its levels with intraocular pressureTzovolou, D. N.; Lamari, F.; Mela, E. K.; Gartaganis, S. P.; Karamanos, N. K.
doi: 10.1002/1099-0801(200008)14:5<301::AID-BMC4>3.0.CO;2-Qpmid: 10960828
The aim of this study was the development of a capillary electrophoretic method for the determination of the levels of the selective alpha2‐adrenergic receptor agonist brimonidine in aqueous humor of the eye and blood sera and their relation to its efficacy in reducing the intraocular pressure (IOP). Analysis of brimonidine was performed by capillary zone electrophoresis using 20 mM borate, pH 9.3, as operating buffer and detection at 255 nm. Brimonidine levels were determined in aqueous humor and blood sera from seven patients admitted for cataract extraction following ocular administration of the ophthalmic Alphagan™ solution. Levels of brimonidine and IOP values were recorded for a 24 h period. Alphagan™ administration resulted in a significant reduction of IOP, from within 30 min up to 4–5 h, whereafter a stepwise increase was recorded until 24 h, where mean IOP value returned to that before administration. The IOP reduction was related to the levels of brimonidine in aqueous humor, where maximal levels (80–100%) were obtained within 1–3 h. A 50% amount of the solution was determined after 4–5 h, whereas it reached the minimum level after 12 h. Serum levels reached maximum within 3–4 h, a 50% reduction was recorded in 12 h and minimum level in 24 h. It is concluded that brimonidine administration may significantly reduce IOP in patients when its level is maintained ≥50% of the maximum present in aqueous humor, i.e within a 4–6 h period. Since at this time the level of brimonidine in blood serum has reached maximum value, administration of brimonidine every 6 h may be used to obtain adequate brimonidine levels to maintain a constantly lowered IOP. Copyright © 2000 John Wiley & Sons, Ltd.
Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high‐performance liquid chromatography and fluorescence detectionSastre Toraño, Javier; Rijn‐Bikker, Petra van; Merkus, Paul; Guchelaar, Henk‐Jan
doi: 10.1002/1099-0801(200008)14:5<306::AID-BMC986>3.0.CO;2-7pmid: 10960829
A validated new and precise reversed‐phase high‐performance liquid chromatographic method for the determination of melatonin in human plasma and cerebrospinal fluid, with 5‐fluorotryptamine as internal standard, is described. Liquid–liquid extraction with dichloromethane was performed under alkaline conditions. After evaporation of the organic solvent, the extract was dissolved in eluent and chromatographed on a base‐deactivated octadecyl column, using an eluent composed of 650 mL potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 350 mL methanol. Fluorescence detection at an excitation wavelength of 224 nm and an emission wavelength of 348 nm was used for quantitation. Melatonin and 5‐fluorotryptamine chromatographed with retention times of 5.3 and 9.3 min, respectively. Mean recoveries of 96% (n = 10) and 95% (n = 5) were found for melatonin in plasma and cerebrospinal fluid respectively. 5‐Fluorotryptamine was found to have a mean recovery of 90% (n = 10) and 82% (n = 5) in plasma and cerebrospinal fluid, respectively. The repeatability coefficients of variation for both melatonin and 5‐fluorotryptamine in plasma were 4–5% (five different samples (r = 5) on two consecutive days (n = 2)), with reproducibility coefficients of 1.6–7% (n = 2, r = 5) and 0.9–4% (n = 2, r = 5) for melatonin and internal standard, respectively. In cerebrospinal fluid the repeatability coefficient of variation of the extraction procedure was 5% (n = 1, r = 5) for melatonin and 7% (n = 1, r = 5) for 5‐fluorotryptamine. The correlation coefficients of the calibration curves were 0.9998 (n = 2) in plasma at a concentration range of 0.108–25.9 ng/mL and 0.9994 (n = 2) at a concentration range of 0.108–25.9 ng/mL in cerebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime. Copyright © 2000 John Wiley & Sons, Ltd.
Post‐column reaction detection of biotin in human plasma ultrafiltrate based on laser‐induced fluorescence energy transfer in the far‐red spectral regionShahdeo, Kamlakshi; Anderson, F. Philip.; Karnes, H. Thomas
doi: 10.1002/1099-0801(200008)14:5<311::AID-BMC988>3.0.CO;2-Fpmid: 10960830
High performance liquid chromatography followed by post‐column reaction detection in the far‐red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on‐line post‐column reaction detection system. The labels used for energy transfer were R‐Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2.44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 × 10−11M) calculated based on a factor of 2× the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates. Copyright © 2000 John Wiley & Sons, Ltd.
Chiral liquid chromatography tandem mass spectrometry in the determination of the configuration of 2‐hydroxyglutaric acid in urineRashed, Mohamed S.; AlAmoudi, Mohamed; Aboul‐Enein, Hassan Y.
doi: 10.1002/1099-0801(200008)14:5<317::AID-BMC989>3.0.CO;2-Vpmid: 10960831
D‐2‐Hydroxyglutaric aciduria and L‐2‐hydroxyglutaric aciduria are two distinct inherited metabolic diseases. The accurate diagnosis of the exact disorder relies on the determination of the configuration of the enantiomers, either D‐2‐hydroxyglutaric acid or L‐2‐hydroxyglutaric acid excreted in excess in urine of patients. The enantiomeric chiral separation of 2‐hydroxyglutaric acid was achieved using a ristocetin A glycopeptide antibiotic silica gel bonded column. The chiral column was interfaced with a tandem mass spectrometer for the purpose of specifically detecting the eluting 2‐hydroxyglutaric acid. Tandem mass spectrometry was employed using an electrospray ion source in the negative ion mode. Three parent‐to‐daughter transitions under collision‐induced dissociation conditions were used to detect only 2‐hydroxyglutaric acid. The two forms of the compound were satisfactorily separated with almost baseline resolution at 4.95 and 5.5 min. Three known patients with 2‐hydroxyglutaric aciduria were identified to have L‐2‐hydroxyglutaric aciduria. The method is simple, selective, rapid, and free from interference. Copyright © 2000 John Wiley & Sons, Ltd.
High‐performance liquid chromatographic assay for the determination of novel triazole antifungal agents in tissue. Application to tissue distribution studiesKhan, Jehangir K.; Montaseri, Hashem; Poglod, Marzena; Bu, Hai‐Zhi; Daneshtalab, Mohsen; Micetich, Ronald G.
doi: 10.1002/1099-0801(200008)14:5<321::AID-BMC990>3.0.CO;2-0pmid: 10960832
A simple and rugged reversed‐phase high‐performance liquid chromatographic method with ultraviolet absorbance detection at 263 nm was developed and validated for the analysis of novel triazole antifungal agents SYN‐2869 and its derivatives in tissues. The method involved homogenization with 0.01 M phosphate buffer (pH 7.8) for lung, brain and spleen tissues. The liver and kidneys were homogenized with acetonitrile:acetone (1:1). The plasma proteins were precipitated with ice‐cold acetonitrile and supernatent was evaporated to dryness. The reconstituted samples were injected onto an HPLC system. SYN‐2869 was separated from the matrix components on a symmetry C18 column using a aqueous mobile phase of acetonitrile and water with a flow rate of 1 mL/min. A step gradient of 40–80% acetonitrile eluted SYN‐2869 and the internal standard (SYN‐2506). The linear range was 0.5–10 µg/g (r2 > 0.99). The limit of quantitation was 0.5 µg/g. The inter‐day precision and accuracy for SYN 2869 standard concentration were from 2.6 to 7.4% and from −1.56 to +3.29%, respectively. The method was applied to tissue samples collected from single intravenous administration to mice to evaluate the distribution of these novel antifungal agents to different tissues. Copyright © 2000 John Wiley & Sons, Ltd.
Determination of morphine 3‐esters in rabbit plasma by high‐performance liquid chromatography with ultraviolet detectionOtter, Karin; Mignat, Christian; Heber, Dieter; Ziegler, Albrecht
doi: 10.1002/1099-0801(200008)14:5<327::AID-BMC991>3.0.CO;2-Gpmid: 10960833
A sensitive high‐performance liquid chromatographic (HPLC) method for the quantitation of the morphine 3‐esters 1(3‐(2,2‐dimethylvaleroyl)‐morphine (A), 3‐(2‐phenylbenzoyl)‐morphine (B) and 3‐(2,2‐diphenylpropionyl)‐morphine (C)) in rabbit plasma is described. Sample preparation was based on reversed‐phase solid‐phase extraction. The compounds were separated on C18 reversed‐phase analytical columns and then determined by ultraviolet detection. The recovery from plasma was 78.7 ± 7.4%, 69.1 ± 6.9% and 75 ± 7.2% (mean ± SD) for A, B, and C, respectively. The present method enabled the detection limit of 0.2, 0.2 and 0.1 ng and quantification limit of 20, 10 and 10 ng/ml for A, B and C, respectively. The developed method was used for determination of the plasmakinetics of these morphine 3‐esters in rabbits. Copyright © 2000 John Wiley & Sons, Ltd.
A new sensitive determination method of estradiol in plasma using peroxyoxalate ester chemiluminescence combined with an HPLC systemYamada, Hiroyuki; Kuwahara, Yasuhiro; Takamatsu, Yasuo; Hayase, Tetsuo
doi: 10.1002/1099-0801(200008)14:5<333::AID-BMC992>3.0.CO;2-Opmid: 10960834
A new sensitive determination method of estradiol in a plasma sample using peroxyoxalate ester chemiluminescence was developed. Estradiol, which was extracted by liquid–liquid extraction using ethyl acetate from plasma, was derivatized with dansyl‐chloride (DNS‐Cl) and separated by reverse‐phase HPLC. The performance of four oxalates, bis(trichlorophenyl)oxalate (TCPO), bis(2,4‐dinitrophenyl)oxalate (DNPO), bis(pentafluorophenyl)oxalate (PFPO), and bis(4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl) oxalate (TDPO), were evaluated using the static system, and DNPO was found to have the most sensitive and stable chemiluminescence at a H2O2 concentration of 30 mM. HPLC‐chemiluminescence system using DNPO for the determination of estradiol was established. The detection limit of dansylated‐estradiol (DNS‐E2) was 15 fmol (4 pg) in the standard solution and 44 fmol (12 pg) in the rat plasma sample at S/N = 3. Copyright © 2000 John Wiley & Sons, Ltd.