bluuebunny/crossref_metadata_2025 huggingface dataset:
Solid phase extraction for an improved assay of physostigmine in biological fluids
Authors: Hurst P. R. , Whelpton R.
Published: May 2005

A simple, selective and very sensitive assay is described for the quantification of physostigmine in blood, plasma and urine. The most appropriate solid phase column was selected after a systematic investigation of nine types of phase. The conditions for solid phase extraction were optimized using [3H]physostigmine so that the overall recoveries were >90%. Physostigmine was retained on alkaline treated cyanopropyl columns and eluted into the minimum volume of methanol, obviating the need for an evaporation step. Extracted samples were quantified by HPLC with a three electrode coulometric detection system. The limit of detection was 50 pg/mL for a 0.5 mL plasma sample. The precision (CV) for 0.5 mL plasma samples containing 50 pg was 8.1%. Application of the method to plasma, blood and urine samples is presented.
Identification by gas chromatography/mass spectrometry of long‐chain fatty acids and alcohols from hamster meibomian glands using picolinyl and nicotinate derivatives
Authors: Harvey D. J.
Published: May 2005

Meibomian secretions from the hamster were hydrolysed with base and examined as TMS, [2H9]TMS, methyl ester/TMS, picolinyl ester/TMS and nicotinate/TMS derivatives by capillary GC/MS. Over 90 compounds, representing over 89% of the hydrolysed fraction, were identified. Fatty acids with chain lengths from 10 to 32 carbon atoms were found, the most common of these were in the C15 to C18 and in the C25 to C30 regions. Chain types were predominantly iso or anteiso branched, mono‐unsaturated (C16 and C18) and straight. Fatty alcohols were mainly from the iso or anteiso series and tended to have longer chain lengths; the major alcohols had anteiso‐25 and 27 and iso‐26‐chains. In these respects the secretions were similar to those reported earlier from other species, although fewer mono‐unsaturated compounds with longer chains (C20 to C30 region) were found than in the rat and human. The steroid fraction was characterized by a larger number of compounds than normally present in secretions of this type. The major compound was cholesterol, in common with that in all other examined species except the rabbit.
Evaluation of pyrimido[5,4‐d]pyrimidine derivatives as peroxyoxalate chemiluminescence reagents using a flow injection system
Authors: Nakashima Kenichiro , Maki Kouichi , Akiyama Shuzo , Imai Kazuhiro
Published: May 2005

Eighteen kinds of pyrimido[5,4‐d]pyrimidines together with several commercially available fluorescent compounds such as perylene, Rhodamine B, etc., were evaluated as the reagents for a peroxyoxalate chemiluminescence (CL) detection system by using a flow injection method. The peroxyoxalate CL reaction employed consists of bis(2,4,6‐trichlorophenyl)oxalate, hydrogen peroxide, triethylamine, and a fluorophore. Under the conditions used, 2,6‐bis[di‐(2‐hydroxyethyl)amino]‐4,8‐dipiperidinopyrimido[5,4‐d]pyrimridine (Dipyridamole) and 2,4,6,8‐tetrathiomorpholinopyrimido[5,4‐d]pyrimidine (1i) gave very intense chemiluminescence intensities which were larger than those of any other commercially available fluorescent compounds tested (e.g., 10 times larger than that of perylene).
Determination of nitrated pyrenes and their derivatives by high performance liquid chromatography with chemiluminescence detection after online electrochemical reduction
Authors: Imaizumi Noriko , Hayakawa Kazuichi , Suzuki Yasuo , Miyazaki Motoichi
Published: May 2005

A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6‐trichlorophenyl)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection.

By the proposed method, nitropyrene, 1,3‐, and 1,8‐dinitropyrenes were detected in sooty emissions of cars after simple purifications.
Determination of fluorescent cyanobenz[f]isoindole derivatives of dopamine and norepinephrine using high performance liquid chromatography with chemiluminescence detection
Authors: Kawasaki Takao , Imai Kazuhiro , Higuchi Takeru , Wong Osborne S.
Published: May 2005

Dopamine (DA), norepinephrine (NE) and 3,4‐dihydroxybenzylamine (DHBA) are converted to highly fluorescent cyanobenz[f]isoindole (CBI) derivatives by reacting these amines with naphthalene‐2,3‐dicarboxaldehyde in the presence of cyanide ion. Femtomole amounts of these CBI derivatives separated by reverse‐phase high performance liquid chromatography can be detected by a post‐column chemiluminescence system utilizing bis(2,4‐dinitrophenyl) oxalate and hydrogen peroxide. Linear detection response was observed between 1 to 600 fmol, giving a signal to noise ratio of approximately 15 at the 1 fmol level. An assay procedure was developed for the determination of DA and NE in 20 μL of urine sample using DHBA as the internal standard.
Identification of 17‐hydroxyprogesterone and other steroid hormones in saliva from a normal child and patients with congenital adrenal hyperplasia by plasmaspray liquid chromatography/mass spectrometry
Authors: Shindo Noriko , Yamauchi Noriko , Murayama Kimie , Fairbrother Ann , Krolik Stefan
Published: May 2005

A very sensitive, selective and simultaneous method for the analysis of salivary steroid hormones was examined by discharge‐assisted thermospray liquid chromatography mass spectrometry (plasmaspray LC/MS). Plasmaspray LC/MS gave [M + M]+ or [MH — H2O]+ as a predominant ion in most of the steroids in this work and in some cases fragment ions were also observed. Eight salivary steroid hormones, pregnenolone, 17‐hydroxypregnenolone, progesterone, 17‐hydroxyprogesterone, 11‐deoxycortisol, cortisol, aldosterone, and testosterone were identified within 10 minutes using the selected ion monitoring technique in conjunction with plasmaspray LC/MS. Progesterone, 17‐hydroxyprogesterone, cortisol, aldosterone and testosterone were detected in 1 mL of saliva from a normal child and two patients with congenital adrenal hyperplasia.
High performance liquid chromatographic assay of Amicar, ε‐aminocaproic acid, in plasma and urine after pre‐column derivatization with o‐phthalaldehyde for fluorescence detection
Authors: Lam Stanley
Published: May 2005

A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 μL of serum or urine sample was added to 10 μL of a 10% w/v zinc sulfate solution and 100 μL of methanol, as previously described (Lam et al., 1980) for the removal of proteins by precipitation. 50 μL of the supernatant was then mixed with 300 μL of 1 M borate buffer containing D‐valine as the internal standard before derivatization with o‐phthalaldehyde. The amino acids were then separated by a stereoselective reversed‐phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L‐proline. The chromatography is highly selective, resolving Amicar from L‐valine which in turn is resolved from its unnatural D‐antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o‐phthalaldehyde derivative by fluorescence.
Estimation of human blood plasma 5‐hydroxyindoleacetic acid and homovanillic acid
Authors: Wright‐Honari S. , Marshall E. F. , Ashton C. H. , Hassanyeh F.
Published: May 2005

A method for simultaneous quantification of plasma homovanillic acid and 5‐hydroxyindoleacetic acid has been developed, permitting more efficient neurochemical examinations of these often interrelated biogenic amine systems. Zinc sulphate and sodium hydroxide solutions were used for precipitating the protein in plasma prior to injection on the column. This technique allows for cleaner chromatography, greater sensitivity and high precision. The method uses high performance liquid chromatographic separations of these compounds on C18 reversed phase columns with electrochemical detection. The detailed results from controls and untreated parasuicide patients are given.
Solid phase extraction and high performance liquid chromatographic determination of doxophylline in plasma
Authors: Lagana A. , Bizzarri M. , Marino A. , Mancini M.
Published: May 2005

A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.
The measurement of nucleoside deaminases by high performance liquid chromatography and their use in clinical chemistry
Authors: Sherwood R. A.
Published: May 2005

The measurement of the nucleoside deaminases—cytidine deaminase, guanosine deaminase and adenosine deaminase—by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre‐eclampsia is probably not helpful.
Quantitation of the enantiomers of rimantadine and its hydroxylated metabolites in human plasma by gas chromatography/mass spectrometry
Authors: Choma Nadia , Davis Preston P. , Edom Richard W. , Fukuda Elaine K.
Published: May 2005

A gas chromatographic/mass spectrometric procedure has been developed for the quantiation in human plasma of the enatiomers of rimanatadine and its three hydroxylated metabolites. The assay utilized derivatization of all analytes with the optically active reagent S‐α‐methyl‐α‐methoxy(pentafluorpheyl)acetic acid, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution techniques. This method has been used to meausure plasma concentrations of the enantiomers of rimantadine, m‐hydroxyrimantadine and p‐hydroxyrimantadine (equatorial and axial epimers) in the ranges 2.5‐250, 2.5‐50, 1.25–62.5 and 1.25–62.5 ng/mL, respectively, in six subjects given a single 200 mg dose of racemic rimantadine. Although there are no significant differences in the concentration‐time profiles of R‐ and S‐rimantadine, large stereospecific differences in the disposition of their metabolites are observed.
Determination of enalapril and its active metabolite enalaprilat in plasma and urine by gas chromatography/mass spectrometry
Authors: Shioya Hiroaki , Shimojo Masako , Kawahara Yukinori
Published: May 2005

The method for the simultaneous determination of angiotensin‐converting enzyme (ACE) inhibitor enalapril and its active metabolite enalaprilat in plasma and urine was developed by gas chromatography/mass spectrometry. Enalapril and enalaprilat in plasma and urine were extracted and cleaned up by using Sep‐Pak C18 and silica cartridges. Derivatization was carried out using diazomethane and trifluoroacetic anhydride. Detection by selected ion monitoring was selected to m/z 288 (enalaprilat) and 302 (enalapril). The detection limit of enalapril and enalaprilat was 200 pg/mL in plasma and 2 ng/mL in urine. This method was applied to the pharmacokinetic analysis of enalapril and enalaprilat in body fluids.
A universal peroxyoxalate‐chemiluminescence detection system for mobile phases of differing pH
Authors: Hayakawa Kazuichi , Minogawa Eriko , Yokoyama Tohru , Miyazaki Motoichi , Imai Kazuhiro
Published: May 2005

A universal peroxyoxalate–chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual‐head short‐stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6‐trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole–nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO–hydrogen peroxide. These two solutions were delivered by the two pump‐heads. After the pH of the column eluate was adjusted to the optimum range (6.5–7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0–8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on‐line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by some modifications of the postcolumn solutions.
Sensitive fluorometric detection of prostaglandins by high performance liquid chromatography after precolumn labelling with 4‐(N,N‐dimethylaminosulphonyl)‐7‐(1‐piperazinyl)‐2,1,3‐benzoxadiazole (DBD‐PZ)
Authors: Toyo'oka Toshimasa , Ishibashi Mumio , Terao Tadao , Imai Kazuhiro
Published: May 2005

A highly sensitive and simple method for the determination of prostaglandins (PGs) by HPLC with fluorescence detection is described. PGs are converted to the corresponding fluorescence derivatives by the reaction with 4‐(N,N‐dimethylaminosulphonyl)‐7‐(1‐piperazinyl)‐2,1,3‐benzoxadiazole (DBD‐PZ) in the presence of 2,2′‐dipyridyl disulphide and triphenyl phosphine in acetonitrile. The reaction is completed at room temperature after 30 min. The DBD derivatives of nine PGs are separated within a single 45 min chromatographic run on a reversed phase ODS column with a linear gradient elution using water and acetonitrile. The detection limits (signal‐to‐noise ratio of 3) calculated from the standard mixture of PGs (6‐keto‐F1α, F1α, F2α, E1, E2, D2, limaprost, A1 and B1) are in the range 1.7–5.0 fmol. The applicability of the proposed procedure is evaluated to the detection of PGs added to rat plasma.
A high performance liquid chromatographic method for the determination of albuterol enantiomers in human serum using solid phase extraction and chemical derivatization
Authors: He Langchong , Stewart James T.
Published: May 2005

A high performance liquid chromatographic method was developed for the simultaneous assay of R(−)‐ and S(+)‐albuterol in human serum. The assay involves solid phase extraction as a sample clean‐up step and derivatization of racemic albuterol to its diastereomeric thioureas with 2,3,4,6‐tetra‐O‐acetyl‐α‐D‐glucopyranosyl isothiocyanate. Chromatographic separation was accomplished under isocratic conditions using an octadecylsilane column and a mobile phase consisting of 29:71 acetonitrile:distilled water containing 0.1% triethylamine, pH 4.0 (adjusted with concentrated phosphoric acid) at a flow rate of 0.8 mL/min. The diastereomers were detected using a fluorescence detector set at 223 nm excitation and no emission filter. Racemic bamethane was used as internal standard. Drug to internal standard peak‐height ratios were linear over a 2–20 ng/mL range for each enantiomer. The limit of detection of each analyte was 1.0 ng/mL (S/N = 3).
High performance liquid chromatographic determination of azelastine and desmethylazelastine in guinea pig plasma and lung tissue
Authors: Langevin C. N. , Pivonka J. , Wichmann J. K. , Kucharczyk N. , Sofia R. D.
Published: May 2005

Analytical methods have been developed for the simultaneous quantitation of azelastine (AZ) and desmethylazelastine (DAZ) in guinea pig plasma and lung tissue. The methods require a 1.00 mL plasma sample and a minimum 0.100 g lung sample. Both methods employ liquid/liquid organic extraction and back‐extraction into dilute acid. Quantitation is performed by high performance liquid chromatography on a 2X250 mm 5 μm HypersilTM CPS column using fluroescence detection. The linear quantitative ranges for AZ.HCI and DAZ.HBr in plasma are 0.156–160 ng/mL and 0.313–160 ng/mL, respectively. The linear quantitative ranges for AZ.HCI and DAZ.HBr in lung tissue are 0.039–20 μg/g for both.
Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column
Authors: McKern Neil M. , Edskes Herman K. , Shukla Dharma D.
Published: May 2005

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C18 columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C18 RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.
Measurement of terbutaline and salbutamol in plasma by high performance liquid chromatography with fluorescence detection
Authors: McCarthy P. T. , Atwal S. , Sykes A. P. , Ayres J. G.
Published: May 2005

A method is described for the determination of terbutaline and salbutamol in plasma from patients given maximal therapy for brittle asthma. The analytes were isolated by solid phase extraction on alkali‐treated Bond‐Elut, unmodified, silica columns and measured by high performance liquid chromatography with fluorescence detection (excitation wavelength 200 nm). The limits of detection for a 1 mL sample containing salbutamol and terbutaline were 1 μg/L and 2.5 μg/L, respectively. The intra‐assay precision (CV) for samples containing 25 μg/L was 3.6 and 5.0% respectively. This method was applied to the measurement of terbutaline in samples from patients given continuous infusions of the drug to assess whether this treatment might result in toxicity.
Analysis of morphine and its 3‐ and 6‐glucuronides by high performance liquid chromatography with fluorimetric detection following solid phase extraction from neonatal plasma
Authors: Hartley R. , Green M. , Quinn M. , Levene M. I.
Published: May 2005

A rapid, sensitive and simple to operate high performance liquid chromatographic method for the simultaneous determination of morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octylsilane bonded silica columns (1 mL Bond Elut C8, 50 mg). Chromatographic separation of morphine and its metabolites was achieved using a mobile phase, consisting of 2 mM sodium dodecyl sulphate in 0.05% phosphoric acid:acetonitrile (71.5:28.5 by volume), at a flow‐rate of 1.2 mL/min, in conjunction with a Waters Nova‐Pak C18 column (300 × 3.9 mm). The analytical column was used in combination with a Guard‐Pak module containing a Nova‐Pak C18 Guard‐Pak insert. Using fluorescence detection (excitation 245 nm, emission 335 nm), plasma levels in the region of 5–10 μg/L for the drug and its metabolites can be detected with only 200 μL of plasma. The method has been applied to studies of the disposition of morphine and its metabolites in premature neonates requiring mechanical ventilation who were receiving the drug intravenously; preliminary findings in patients at steady state are presented.
Direct enantiomeric high performance liquid chromatographic separation of propafenone and its major metabolite in serum on a cellulose tris‐3,5‐dimethylphenyl carbamate chiral stationary phase
Authors: Aboul‐Enein Hassan Y. , Bakr Soliman A.
Published: May 2005

A direct, isocratic and simple liquid chromatographic method is described for the enantiomeric separation of propafenone (PRO) and its major metabolite, 5‐hydroxypropafenone, using a cellulose tris‐3,5‐dimethylphenyl carbamate (Chiralcel ODS) column. The sterochemical separation factor (α) obtained was 1.14 and the maximum stereochemical resolution factor (R) was 0.80 when using a mobile phase consisting of hexane: 2‐propanol: diethylamine (90:10:0.4) at 23°C. The hydroxy metabolite could not be successfully separted into its corresponding enantiomers under these chromatographic conditions. The method has been used to determine and identify the enantiomers of PRO and its hydroxy metabolite in a serum sample.
Determination of 5,10,15,20‐ tetra‐ (m‐hydroxyphenyl)chlorin in human plasma by high performance liquid chromatography
Authors: Wang Qiang , Ris Hans‐Beat , Altermatt H. J. , Reynolds Brenda , Stewart J. C. M. , Bonnett Raymond , Lim Chang Kee
Published: May 2005

A high performance liquid chromatographic method for the determination of the photodynamic chemotherapeutic agent 5,10,15,20‐tetra(m‐hydroxyphenyl)chlorin (m‐THPC) in human plasma following intravenous infusion is described. The procedure involves extraction of the drug in plasma with methanol/dimethyl sulphoxide (4:1 v/v) containing 5,10,15,20‐tetra(p‐hydroxyphenyl)clorin as the internal standard and separation on a C18 reversed phase column with acetonitrile:0.1% trifluoroacetic acid (77:23 v/v) as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 423 nm with a detection limit of 15 ng/mL (signal‐to‐noise ratio of 5). The intra‐ and inter‐assay coefficients of variation (CV) on analysis of a plasma spiked with m‐THPC (1 μg/mL) were 2.3 and 3.4% (n=6), respectively.
Synthesis of novel fluorogenic edman reagents, 7‐N,N‐dimethylaminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl)isothiocyanate (DBD‐NCS) and 7‐aminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl)‐isothiocyanate (ABD‐NCS)
Authors: Imai Kazuhiro , Uzu Sonoko , Nakashima Kenichiro , Akiyama Shuzo
Published: May 2005

Novel fluorogenic Edman reagents, 7‐N,N‐dimethylaminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl)isothiocyanate (DBD‐NCS) and 7‐aminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl)isothiocyanate (ABD‐NCS) having no fluorescence themselves were synthesized. The derivatives of amino acids with DBD‐NCS fluoresce at 505 nm with excitation at 385 nm. They were separated on a reversed‐phase HPLC column and detected at the sub‐picomol level. Ala‐Phe derivatized with DBD‐NCS was cleaved by acid to generate DBD‐thiocarbamyl‐Ala.
Determination of paracetamol (acetaminophen) in blood and plasma using high performance liquid chromatography with dual electrode coulometric quantification in the redox mode
Authors: Whelpton Robin , Fernandes Karin , Wilkinson Kathleen A. , Goldhill David R.
Published: May 2005

A simple, but sensitive, micro‐assay for paracetamol in blood and plasma is described. Samples were prepared by precipitation with trichloroacetic assay solution and the drug quantified by reversed phase liquid chromatography with dual electrode electrochemical detection in the redox mode (oxidation at +0.25 V followed by reduction at −0.15 V). The limit of detection was 0.1 mg/L for a 10 μL sample of blood or plasma, the coefficient of variation at this concentration being 11.4%. The method was applied to a study of gastric emptying in patients before and after cardiac surgery.
The determination of a new inhalational anaesthetic, sevoflurane, using an internal standard, xenon, by gas chromatography/mass spectrometry/selected ion monitoring
Authors: Miyano Kazuko , Tanifuji Yasumasa , Obata Toru
Published: May 2005

A novel determination method for a new inhalation anaesthetic, sevoflurane, in a closed circuit in the presence of carbon dioxide absorbents is investigated using gas chromatography/selected ion monitoring with xenon as an internal standard. The decrease rate of sevoflurane with soda lime was 0.22%±0.158/h (mean±SD), while that with baralyme was 0.57%±0.115/h (mean±SD).
Determination of rafoxanide and closantel in ovine plasma by high performance liquid chromatography
Authors: Benchaoui H. A. , McKellar Q. A.
Published: May 2005

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.
Analysis of free and bound O‐phosphoamino acids in urine by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Nakai Kiyohiko , Katagiri Yoshimi , Makita Masami
Published: May 2005

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.
Gas chromatographic determination of mexiletine in human plasma with flame ionization detection after reaction with carbon disulphide
Authors: Song‐gang Ji , Qing‐hong Kong , Xiu‐lu Li , Ping Li
Published: May 2005

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.
Simple analysis of amoxycillin in plasma by high performance liquid chromatography with internal standardization and ultraviolet detection
Authors: Charles Bruce , Chulavatnatol Suvatna
Published: May 2005

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.
Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography
Authors: Castagnola Massimo , Di Pierro Donato , Scatena Roberto , Tavazzi Barbara , Nocca Giuseppina , Rossetti Diana Valeria , Giardina Bruno
Published: May 2005

The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.
High performance liquid chromatographic determination of levomepromazine in human breast milk and serum using solid phase extraction
Authors: Ohkubo Tadashi , Shimoyama Ritsuko , Sugawara Kazunobu
Published: May 2005

A high performance liquid chromatographic (HPLC) method has been developed for the determination of levomepromazine in human breast milk and serum. The levomepromazine was extracted by a rapid and simple extraction method using a Sep‐Pak C18 cartridge. The extracts were separated by HPLC on a C8 bonded reversed phase column and detected by UV absorbance at 254 nm. There was no interference with endogenous substance in human breast milk and serum. A linear relationship was obtained for the levomepromazine over the concentration range of 10–300 ng/mL. The recoveries of levomepromazine added to human breast milk and serum were 92.5–99.1% and 86.9–103.9%, respectively.
Chromatographic determination method for 1‐nitropyrene and its metabolites in biological samples with fluorescence detection after on‐line reduction
Authors: Hayakawa Kazuichi , Terai Noriko , Suzuki Kumiko , Dinning Paul G. , Yamada Masafumi , Miyazaki Motoichi
Published: May 2005

A high performance liquid chromatographic (HPLC) method with fluorescence detection after on‐line reduction for the determination of 1‐nitropyrene (NP), 1‐nitrosopyrene (NSP), 1‐aminopyrene (AP) and N‐acetylaminopyrene (AAP) has been developed. The reduction efficiency of NP and NSP on a zinc column was found to be higher than that of an electrochemical reducer. Using a HPLC equipped with a zinc column (4.0 mm i.d. × 10 mm) and an imidazole/HCIO4 (pH 6.8):acetonitrile mobile phase, detection limits (S/N = 3) of 20‐30 fmol for NP, NSP and AP and 350 fmol for AAP were obtained. NP, NSP and AP were determined in the incubation mixture of NP and Salmonella typhimurium, YG1021, by this method. Time course studies showed that a large ratio of NP was metabolized in the pre‐incubation step of the Ames test.
Derivatization with fluorogenic benzofurazan reagents of amino acid enantiomers and their separation on a pirkle column
Authors: Imai Kazuhiro , Fukushima Takeshi
Published: May 2005

L‐ and D‐Amino acids (Leu or Phe) were derivatized with fluorogenic reagents, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), 4‐aminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole (ABD‐F) and 5‐(N,N‐dimethylamino)naphthalene‐1‐sulfonylchloride (DNS‐Cl), and separated on a Pirkle type column, Sumichiral OA 2500 (S) ((S)‐1‐naphthylglycyl‐3,5‐dinitrophenylamide silica gel) with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection of the derivatives was made at 530 nm, 590 nm and 530 nm with excitation at 470 nm, 450 nm, 450 nm and 350 nm, respectively. The former three derivatives of the enantiomers were separated well from each other; The as for each NBD‐, DBD‐ and ABD‐derivative of L‐ and D‐Leu were 1.10, 1.11 and 1.10, respectively. However, the DNS derivatives of L‐and D‐Leu were not separated (separation factor, α = 1.0). All NBD‐ and ABD‐derivatives of L‐ and D‐Phe were also well separated (αs were 1.18, 1.17 and 1.16, respectively), while DNS‐L‐ and ‐D‐Phe were barely separated (α = 1.04). These data suggest that the 2,1,3‐benzoxadiazole (benzofurazan) moiety is very effective and preferable to the dimethylaminonaphthalene sulfonyl (DNS) structure for the separation of enantiomers of amino acids derivatized with benzofurazan reagents.

No recemization of each enantiomer occurred during the derivatization reaction with NBD‐F, DBD‐F and ABD‐F at pH 8.0 and 60°C for 2 min, at pH 9.3 and 60°C for 30 min and pH 9.3 and 60°C for 60 min, respectively, meaning that all benzofurazan type fluorogenic reagents are useful for sensitive determination of amino acid enantiomers.
Enantioselective bioanalysis of beta‐blocking agents: Focus on atenolol, betaxolol, carvedilol, metoprolol, pindolol, propranolol and sotalol
Authors: Egginger Gabriele , Lindner Wolfgang , Vandenbosch Christel , Massart Desire Luc
Published: May 2005

The recent developments in enantioselective HPLC‐separation techniques are impressive and are driven by industrial and academic interests; thus there is for instance a high demand for developing stereoselective assays for chiral drugs in biological fluids. The beta‐blocking agents, which possess an amino‐ propanol‐ or ‐ethanol side chain with at least one chiral centre, represent one of the most intensively investigated groups of more than 40 drugs introduced world wide. Seven of the most popular beta‐blockers were chosen as representatives: atenolol; betaxolol; carvedilol; metoprolol; pindolol; propranolol; and sotalol, these span the whole range of lipophilicity to hydrophilicity (polarity). Enantioselective HPLC bioassays for these β‐blockers published so far, including techniques based on chiral derivatizing agents (CDAs), chiral stationary phases (CSPs) and chiral mobile phase additives (CMPAs) have been reviewed and documented in the light of general aspects together with pharmacokinetic and pharmacodynamic considerations.
High performance liquid chromatography of tamoxifen and metabolites in plasma and tissues
Authors: Lim C. K. , Chow Loretta C. L. , Yuan Zhi‐Xin , Smith L. L.
Published: May 2005

An isocratic reversed‐phase high performance liquid chromatographic method for the determination of tamoxifen and its metabolites in plasma and tissues is described. Plasma or tissue homogenate was extracted with methanol/dimethyl sulphoxide (4:1 v/v). The supernatant after centrifugation was separated on a BDS‐Hypersil column with methanol/0.5 M ammonium acetate (75:25 v/v) as the mobile phase. The recoveries of tamoxifen added to plasma and liver tissue homogenate by the extraction procedure were 102± 1.6 and 98±2.4% (mean±SD, n = 6), respectively. The solutes were detected at 280 nm with a detection limit of 0.25 μg/mL for tamoxifen.
Relations between blood pressure and plasma norepinephrine concentrations after administration of diltiazem to rats: HPLC‐peroxyoxalate chemiluminescence determination on an individual basis
Authors: Higashidate Sakae , Imai Kazuhiro , Prados Pablo , Adachi‐Akahane Satomi , Nagao Taku
Published: May 2005

A calcium antagonist, diltiazem, was infused continuously into Sprague‐Dawley rats through the left femoral vein at four different flow rates. The mean arterial blood pressure and concentrations of plasma norepinephrine (NE) were measured in each single rat (n = 5) and the correlations between them were studied. Blood (150 μL) was collected 13 times during the infusion. Plasma NE was determined by HPLC–ethylenediamine condensation reaction–peroxyoxalate chemiluminescence detection system (HPLC–ED–PO‐CL).

In four cases from 5 rats, the blood pressure reduction caused by diltiazem was inversely correlated to logarithm of plasma NE concentration. The relation was expressed as Y = −α logX + m. The coefficients of correlation were −0.9506, −0.9293, −0.9341 and −0.8675, respectively. The correlation for the last rat was worse (r = −0.0799). The good correlation would imply that the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the blood pressure reduction caused by diltiazem.

The present experiment proved the feasibility of the determination method of NE utilizing HPLC–ED–PO‐CL detection in applying to the individual rats.
Identification and verification of the anabolic steroid boldenone in equine blood and urine by HPLC/ELIS
Authors: Hagedorn Heinz‐Werner , Schulz Rüdiger , Jaeschke Gerhard
Published: May 2005

An enzyme linked immunosorbent assay (ELISA) was developed to detect the anabolic steroid boldenone in equine blood and urine. The polyclonal antiserum was raised in rabbits, employing boldenone‐17‐hemisuccinate–bovine serum albumin as antigen. Boldenone‐17‐hemisuccinate–horseradish peroxidase served as enzyme conjugate. Sensitivity of the assay was 26.0 ± 3.0 pg/well. Among the endogenous steroids tested only progesterone and testosterone exhibited moderate cross‐reactivities, 3.4 and 2.5%, respectively. These crossreactivities are of no importance for the boldenone assay. For the reduction of background levels, srceening for boldenone of equine serum was performed after extraction. Urine samples were determined directly after dilution, omitting hydrolysis of boldenone conjugates. Positive screening results were confirmed by means of two independent HPLC systems combined with off‐line detection, employing the boldenone ELISA. Methandienone served as internal standard to ascertain retention factors. In horses treated with boldenone‐17‐undecylenate the presence of boldenone in serum was confirmed up to 28 days and in unhydrolysed urine up to 56 dayspost applicationem.
Quantitative relationships between the structure of β‐adrenolytic and antihistamine drugs and their retention on an α1‐acid glycoprotein HPLC column
Authors: Nasal Antoni , Radwańska Aleksandra , Ośmiałowski Krzysztof , Buciński Adam , Kaliszan Roman , Barker Geoffrey E. , Sun Peng , Hartwick Richard A.
Published: May 2005

Chromatographic retention parameters of a series of 7 β‐adrenolytics and of 12 antihistamine drugs were determined employing an α1‐acid glycoprotein (AGP) high‐performance liquid chromatographic (HPLC) column. For the group of antihistamines capillary electrophoretic (CE) retention was additionally measured in the presence of either AGP or human serum albumin (HSA). Two series of solutes hydrophobicity parameters were obtained by reversed‐phase HPLC on an immobilized artificial membrane (IAM) column. The solutes studied were subjected to molecular modelling and the structural descriptors obtained were applied in studies of quantitative structure‐retention (protein binding) relationships (QSRR). It was found that retention on AGP correlates well with the literature on physiological protein binding data. This retention was demonstrated to depend on hydrophobicity: to a lesser extent in the case of β‐adrenolytics and strongly in the case of antihistamines. Hydrophobicity, along with molecular width and electron excess charge on aliphatic nitrogen was demonstrated to describe retention of antihistamines on AGP. The AGP column is recommended as a convenient reactor for studies of drug–protein interactions. Preliminary CE data do not correlate with the HPLC data.
Proteinergic profiles in cerebrospinal fluid from alcoholic subjects
Authors: Silberring Jerzy , Brostedt Peter , Neiman Jack , Hellman Ulf , Liljequist Sture , Terenius Lars
Published: May 2005

Minute amounts of cerebrospinal fluid samples from alcoholics were subjected to separation by HPLC‐molecular sieving, combined with multispectral UV analysis of the acquired data. A significant difference in the protein/polypeptide pattern within the molecular weight range of 7–10 kDa has been observed between samples, taken directly after detoxification and 2 weeks later. Spectral analysis of the results suggests that the components are of peptidergic nature. On the other hand, albumin content did not differ significantly, suggesting that the blood–brain barrier was not affected. An enzyme marker, dynorphin converting enzyme, remained unchanged in both groups.
Enantiospecific determination of ibuprofen in rat plasma using chiral fluorescence derivatization reagent, (−)‐2‐[4‐(1‐aminoethyl)‐phenyl]‐6‐methoxybenzoxazole
Authors: Kondo Junichi , Suzuki Nobuyuki , Naganuma Hideo , Imaoka Toshi , Kawasaki Takao , Nakanishi Akio , Kawahara Yukinori
Published: May 2005

A highly sensitive and reversed‐phase high‐performance liquid chromatographic method for the determination of the enantiomeric composition of ibuprofen in rat plasma is described. The method is based on the resolution of the diastereomeric amides formed on reaction of the ibuprofen enantiomers with (−)‐2‐[4‐(1‐aminoethyl)phenyl]‐6‐methoxybenzoxazole ((−)‐APMB) in the presence of 2,2′‐dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) in dichloromethane. The reaction mixture was allowed to stand at room temperature for 5 min, and the reaction was completed by evaporation with a stream of nitrogen at 40°C. The minimum quantifiable concentrations were 0.2 μg/mL and 0.4 μg/mL for S‐ibuprofen and R‐ibuprofen, respectively, in a 10 μL injection volume. The method was applied to the determination of enantiomeric ibuprofen in plasma after oral administration to rats.
Gas chromatography–mass spectrometry determination of etodolac in human plasma following single epicutaneous administration
Authors: Giachetti Claudio , Assandri Alessandro , Zanolo Giovanni , Brembilla Emilio
Published: May 2005

A highly sensitive gas chromatographic–mass spectrometric method for the determination of etodolic acid, as methyl ester, in plasma was developed. The feasibility and specificity of the method was ascertained monitoring the concentration levels in plasma samples collected from 12 male healthy volunteers given epicutaneously 5 g of 10% etodolac gel formulation.
TLC—A rapid method for liposome characterization
Authors: Brailoiu Eugen , Saila Liliana , Huhurez Gabi , Costuleanu Marcel , Filipeanu Catalin M. , Slatineanu Sebastian , Cotrutz Carmen , Branisteanu Dimitrie D.
Published: May 2005

One of the most important problems for the use of liposomes as a drug delivery system is the modification of the vesicle induced by the liquid medium in which they are introduced (blood plasma for in vivo studies and the saline buffer solution for in vitro studies).

Using thin‐layer chromatography (TLC) we compared the behaviour of phosphatidylcholine (used for liposomes preparation) to that of the following unfilled liposomes: multilamellar liposomes (MLV); small unilamellar vesicles (SUV); and reverse phase evaporation vesicles (REV), before and after storage for 15 min in Krebs‐Henseleit solution (37°C, pH 7.4, aereated continuously with 95% O2 + 5% CO2). All variants contained the same amount of phosphatidylcholine.

Thin‐layer chromatography was performed on silica gel 60 as adsorbant. Two types of solvents were tested: one based on chloroform/alcohol (n‐butanol or n‐propanol or methanol)/water mixture (in different ratios) and another based on alcohol/alcohol/water mixture (n‐butanol/n‐propanol/water in 4/3/3 volume ratio). In all variants of chloroform containing solvents no differences were found between phosphatidylcholine and all types of liposomes.

When using as solvent n‐butanol/n‐propanol/water significant differences were found between all types of liposomes before and after storage in Krebs‐Henseleit solution. Their presence, after TLC treatment, was shown in electron microscopy studies.
Separation of vitamin B complex and folic acid using TLC plates impregnated with some transition metal ions
Authors: Bhushan Ravi , Parshad Vineeta
Published: May 2005

The separation of Vitamin B complex and folic acid was achieved on silica gel layers impregnated with Mn++, Fe++, Co++, Ni++, Cu++, Cd++, Zn++ or Mg++ (0.1%, 0.2%, 0.3%, 0.4% of each ion). Three new solvent systems n‐propanol: n‐butanol: water: ammonia (7:5:1:2, by vol), n‐propanol: n‐butanol: water: ammonia (7:5:1:1.5, by vol), n‐propanol: n‐butanol: water: ammonia (7:5:0.75:2, by vol) were developed. The spots of vitamins were located by exposing the chromatoplate to iodine vapours.
A non‐radioactive iothalamate and p‐aminohippuric acid high‐performance liquid chromatographic method for simultaneously measuring glomerular filtration rate and renal blood flow in the rat
Authors: Bell Rosonald R. , Bombardt Paul A. , DuCharme Donald W. , Kolaja Gerald J. , Packwood William H. , Bothwell Brian E. , Satoh Paul S.
Published: May 2005

A high‐performance liquid chromatographic (HPLC) assay method has been developed for the quantitative determination of iothalamate and p‐aminohippuric acid (PAH) concentrations in serum and urine samples in the male rat. Glomerular filtration rate (GFR) was measured as clearance of iothalamate, while effective renal blood flow (ERBF) was measured as clearance of PAH. The method is simple, rapid and sensitive and detects iothalamate and PAH in rat serum and urine following administration of bolus doses and continuous infusions of iothalamate and PAH. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, and an aliquot chromatographed on a C18 reversed‐phase column. The mobile phase was comprised of 0.1 M sodium phosphate with 1.2 mM tetrabutylammonium phosphate: methanol, 85:15 (v/v), at a flow rate of 1.0 mL/min. The analytical column eluate was monitored with a UV detector at 254 nm with quantitation achieved using peak‐height ratios. The precision of the method was 6.6 and 3.6% for iothalamate in serum and urine, and 5.6 and 4.9% for PAH in serum and urine, respectively. The lower limit of quantitation was 0.63 μg/mL for iothalamate and 1.25 μg/mL for PAH in serum, and 3.1 μg/mL for iothalamate and 1.5 μg/mL for PAH in urine. Recovery of iothalamate from serum and urine was 99.9 and 93.5%, respectively. Recovery of PAH from serum and urine was 99.8 and 92.6%, respectively.

The present study demonstrated that non‐radioactive iothalamate and PAH can be measured simultaneously using a HPLC assay to measure GFR and ERBF in the male rat.