A high-performance liquid chromatographic assay with improved selectivity for cisplatin and active platinum (II) complexes in plasma ultrafiltrate

cis-Diamminedichloroplatinum(II) (DDP) was measured in plasma ultrafiltrate following derivatization with sodium diethyldithiocarbamate (DDTC) by quantitation against a nickel chloride internal standard. A chloroform extract containing the Pt(DDTC)2 and Ni(DDTC)2 complexes was separated by reversed-phase high-performance liquid chromatography on a C18 radial compression column. The complex was eluted with methanol/water, 4/1, at a flow rate of 1.5 ml/min, and was detected at 254 nm. The limit of sensitivity was 0.1 microgram/ml DDP in the ultrafiltrate. This analytical approach was validated by comparison to graphite furnace atomic absorption spectrophotometric determinations of duplicate samples. There was clearly a component of the ultrafiltrable platinum present that was resistant to derivatization by DDTC. Evidence is presented that this component, presumably Pt(II) complexed with endogenous small molecules, is non cytotoxic and, hence, that this method may be selective for "active Pt(II)." This method offers an advantage over atomic absorption determination of total platinum in ultrafiltrate which does not discriminate between active and inactive forms, and over off-line FAA detection of parent DDP in HPLC eluates which ignores other active forms. Using this technique we have measured the pharmacokinetics of DDTC-reactive Pt(II) in humans after either i.v. infusion or infusion of DDP into the peritoneal cavity of patients with ovarian carcinoma.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Cycloheximide-sensitive recovery from 254 nm UV light damage in cultured marsupial cells

Colony-forming ability of Potorous tridactylus-kidney (PtK-2) cells was measured after exposure to 254-nm ultraviolet (UV) light and cycloheximide. Addition of 5 microM cycloheximide after exposure of the cells to UV light greatly decreased cell survival. Maximum effect of the drug was obtained by 24-h exposure after irradiation. The cycloheximide sensitivity of irradiated cells was eliminated if addition of the drug was delayed for 8-10 h after irradiation, or if the cells received photoreactivating light treatment prior to cycloheximide exposure. Thus, a major component of pyrimidine dimer removal may involve a cycloheximide-sensitive mechanism.     

Simultaneous determination of three carbapenem antibiotics in plasma by HPLC with ultraviolet detection

A simple, precise and accurate high-performance liquid chromatography (HPLC) method using ultraviolet (UV) detection has been developed for simultaneous determination of carbapenem antibiotics: imipenem, meropenem and ertapenem in human plasma. Samples were spiked with ceftazidime as internal standard and proteins were precipitated by acetonitrile. Separation was achieved on a C8 column with a mobile phase composed of phosphate buffer 0.1M (pH 6.8) and methanol in gradient elution mode. Detection was performed at 298 nm. Calibration curves were linear from 0.5 to 80 mg/L for each compound, with correlation coefficients over 0.997. Intra- and inter-day validation studies showed accuracy between -4.5 and 8.1% and precision below 10.4%. Mean recoveries were 82.2, 90.8 and 87.7% for imipenem, meropenem and ertapenem, respectively. This method provides a useful tool for the therapeutic drug monitoring of carbapenems.     

Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-¹⁴C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.     

Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.     

Determination of amino acids in fodders and raw materials using capillary zone electrophoresis

Two schemes were offered for analysis of amino acid contents in fodders and raw materials for mixed fodders by capillary zone electrophoresis (CZE). The first variant provides express analysis of four technologically important amino acids (lysine, methionine, threonine, cystine) in borate buffer on characteristic absorption of aminogroup (190 nm), with limits of quantitation being on average 0.2%. The second scheme includes pre-capillary derivatization of amino acids using phenylisothiocyanate (PITC) and separation of phenylthiocarbamyl (PTC)-derivatives obtained by CZE with a detection on 254 nm, which allows to widen a list of detectable components up to 19 (without tryptophan) and significantly improve detection limits down to 0.01%. Acid hydrolysis was used for a sample preparation. The results of analysis of fodders were compared using such methods, as CZE, ion exchange chromatography (amino acid analyzer) and reversed-phase (RP)-HPLC (with gradient technique of elution).     

Development of an HPLC assay for the analysis of tetrafluoroputrescine--a putrescine analog

A simple reverse-phase liquid chromatographic system with ultraviolet detection at 254 nm for tetrafluoroputrescine (TFP), a putrescine analog, is described. The assay involves precolumn derivatization of TFP with dansyl chloride at pH 6.8-7.5 at 60 degrees C, followed by separation from putrescine (PUT) and quantitation. The derivatization procedure was adapted for the simultaneous analysis of TFP and PUT in whole blood components. Also, preliminary studies on protein binding of TFP are reported.     

High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture

In this work, we present an improved method for the determination of a wide range of intracellular metabolites from mammalian cells by anion-exchange chromatography. The analysis includes the measurement of intermediates from glycolysis and tricarboxylic acid cycle as well as several additional nucleotides and sugar nucleotides. The use of an electrolytic on-line eluent generation device made the method highly convenient, reliable and prone to errors. Due to short delay times of the eluent generator, rapid KOH gradient changes could be applied to improve separation and to speed up elution. Suppressed conductivity and UV in series was used for detection. The detection wavelength of the UV detector was switched from 220 to 260 nm during the elution for a more selective signal depending on the absorption of analytes. Standards from more than 50 metabolites of major cellular pathways were chromatographically tested and compared to chromatograms from extraction samples of Madin-Darby canine kidney (MDCK) and BHK21 cells. A validation for most substances was performed. Detection limits were below the micromolar range and the coefficient of correlation (R(2)) was above 0.99 for most analytes. Working ranges were between 0.125-3.875 and 4.5-139.5 microM. Sample pH had a major impact on the quantification of several metabolites, but measurements were robust within a pH range of 6.5-9.0.     

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.     

Action of UV radiation on the surface of mammalian immunocompetent cells. III. Ultrastructural changes in the glycocalyx of mouse lymphocytes

The outer perimembrane layer of murine spleen lymphocytes were studied with electron microscopy. Mice were treated with short-wave (254 nm) and long-wave (365 nm) ultraviolet radiation in isoeffective lethal doses. The other perimembrane layers were stained with Alcian blue and Ruthenium red. The 254 nm UV treatment decreased the level of dye--sorption by these perimembrane layers, whereas no such effect was obvious after the 365 nm UV treatment.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

Ultrasensitive capillary electrophoresis of sulfated disaccharides in chondroitin/dermatan sulfates by laser-induced fluorescence after derivatization with 2-aminoacridone

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived Δ-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated Δ-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized Δ-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.     

Measurement of 5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol) in human plasma and urine by high-performance liquid chromatography

Three high-performance liquid chromatographic methods are described for the detection of the novel antifolate anticancer drug (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol): one with fluorometric detection and two with detection by UV absorbance. An assay for plasma lometrexol using UV detection (288 nm) and reversed-phase chromatography was developed, with a quantitation limit of 0.2 μg/ml and linearity up to 10 μg/ml. This assay was modified for measurement of lometrexol in urine, with a quantitation limit of 2 μg/ml and linearity up to 25 μg/ml. An alternative assay for plasma lometrexol using derivatization and fluorescence detection (excitation at 325 nm, emission at 450 nm) was also developed, which proved twenty-fold more sensitive (quantitation limit of 10 ng/ml) than the UV assay, and which was linear up to 250 ng/ml. The fluoremetric method requires sample oxidation with manganese dioxide prior to analysis, and uses ion-pair chromatography with tetramethylammonium hydrogensulphate as an ion-pair reagent. All assays use a similar preliminary solid-phase extraction method (recovery as assessed by UV absorption >73%), with C10-desmethylene lometrexol added for internal standardisation. Each assay is highly reproducible (inter-assay precision in each assay is <10%). Applicability of the fluorescence-based assay to lometrexol in plasma and the UV-based assay lometrexol in urine is demonstrated by pharmacokinetic studies in patients treated as part of a Phase I clinical evaluation of the drug.     

Design of a UV-C irradiation process for the inactivation of viruses in protein solutions.

The ability of ultraviolet (UV) light to inactivate viruses is well established. However, attempts to apply this to the manufacture of pharmaceutical proteins have been limited by incomplete treatment, low capacity or excessive dilution. Effective processing of large-scale batches of UV-opaque protein solutions has been achieved using a continuous-flow device. The operation of this device has been modelled and a design equation derived to relate the processing conditions and product characteristics to the degree of virus inactivation obtained. Variables included in the model are UV-absorbance at 254 nm (A(254)), hydrodynamic properties of the protein solution, residence time, intensity of UV light and diameter and length of irradiation tube. With this information a specific constant was calculated for each virus which denotes its relative sensitivity to UV and from which the degree of virus inactivation expected can be estimated.     

Determination of cisplatin and cis-diammineaquachloroplatinum(II) ion by liquid chromatography using post-column derivatization with diethyldithiocarbamate

A post-column derivatization method has been developed for the determination of cisplatin and its monohydrated form. Cisplatin was isolated on a strong anion-exchange column, while a strong cation-exchange column was used for the monohydrated complex. Diethyldithiocarbamate was used as reagent and the influence of temperature, pH and methanol content on the yield of derivative was investigated. The reaction was quantitative using a packed-bed reactor with a surrounding temperature of 115°C and a mobile phase consisting of 0.125 M succinic acid—sodium hydroxide buffer pH 5.2 and methanol (2:3, v/v). The resulting complex, Pt(DDTC)₂, was monitored photometrically at 344 nm. The precision of the determination was 11.5% (C.V.) at an injected amount of 20 ng (n = 12) for monoaqua and 8.0% (C.V.) at 9 ng (n = 10) for cisplatin. The method was used to evaluate the plasma concentration of cisplatin and its monohydrated form in a patient.     

Simultaneous determination of betamethasone,betamethasone acetate and hydrocortisone in biological fluids using high-performance liquid chromatography

A sensitive, specific, reproducible and rapid high-performance liquid chromatographic method for the simultaneous quantitation of betamethasone, betamethasone 21-acetate and hydrocortisone in biological fluids is described. Hydrocortisone acetate is used as an internal standard and the samples are extracted with dichloromethane before chromatographing on a reversed-phase system. Detection at two ultraviolet wavelengths (254 nm and 240 nm) was used to assess the specificity of the system, and the sensitivity was found to be greater than 10 ng for all steroids. The speed with which this assay can be performed makes it particularly useful for pharmacokinetic studies, and plasma concentration—time profiles resulting from the administration of betamethasone phosphate and betamethasone acetate are presented.     

Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100mM boric acid buffer containing 10mM SDS and 10mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0 x 10(-7) to 9.0 x 10(-7) M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.     

粪臭素对胃蛋白酶部分理化性质的影响

采用紫外光谱法和荧光光谱法研究了粪臭素与胃蛋白酶的结合作用。观测到粪臭素使胃蛋白酶的紫外吸收峰增强,特征荧光峰淬灭。Stern-Volmer淬灭曲线显示,粪臭素对胃蛋白酶的荧光淬灭很可能是一个单一的静态淬灭过程。