Novel assay method for mitoxantrone in plasma, and its application in cancer patients

Mitoxantrone is an anthracene derivative that acts as a cytostatic in a variety of cancers. A quantitative analytical method has been established for the determination of mitoxantrone in plasma. The method employed C18 reversed-phase ion-pair chromatography with an isocratic mobile phase of 50.0% methanol in 10 mM phosphate buffer (pH 3.0) plus 0.09% 1-pentanesulphonic acid and ultraviolet detection. Sample preparation consisted of two extraction steps using same organic solvent system at different pH to remove plasma impurities efficiently. Potential adsorption of mitoxantrone onto glassware was considered. Silanization of all glassware with 5% dichlorodimethylsilane in chloroform increased the extraction recovery in plasma from 50 to 85% with high reproducibility. Mitoxantrone was unstable in human plasma. To maintain plasma sample integrity, each millilitre of sample should be fortified with 0.1 ml of 5% vitamin C (in citrate buffer) and kept frozen until analysis. Using this new method, the calibration curve of mitoxantrone in plasma in the range of interest (1-500 ng/ml) showed good linearity (r = 0.996) and precision (both between-day and within-day coefficients of variation less than 10%). The lower detection limit of this assay method was 1 ng. The application of this method allowed us to study the stability of mitoxantrone in plasma, and the pharmacokinetics of mitoxantrone in nasopharyngeal carcinoma patients receiving 12 mg/m2. The study revealed a prolonged terminal phase half-life for mitoxantrone.     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

A novel separation and preconcentration method for traces of manganese, cobalt, zinc and cadmium using coagulation of colloidal silica.

A separation and preconcentration method has been developed for traces of heavy metals using coagulation of colloidal silica. An appropriate amount of colloidal silica was added to a sample solution, the pH was adjusted to 11 with tetramethylammonium hydroxide solution and calcium chloride solution was then added to coagulate the silica. The coagulated silica and solution were separated by centrifugation, and the silica was then treated with hydrofluoric and perchloric acids. The residue was taken up in dilute nitric acid and subjected to ICP-AES to determine manganese, cobalt, zinc and cadmium. The proposed method was successfully applied to analysis of river-water.     

HPLC Silica Column Fractionation of Pesticides and PCB from Butterfat

Abstract

Separation of trace levels of toxic pesticides and other chemical contaminants (e.g., PCB) from edible fats and oils in preparation for quantitation by gas chromatography (i.e., GC-EC, GC-MS, etc.) continues to be an important analytical concern. Commercial HPLC silica columns were evaluated for practical application to the fractionation of selected compounds of different polarity from butterfat. Samples were injected in hexane solution and fractionated using isocratic hexanemethylene chloride mobile phases. Fat was readily purged from the columns with methylene chloride. A 254-nm UV detector was used to monitor elution patterns of concentrated standard solutions and of lipid material. Analyte fractions subsequently collected from samples fortified at sub-ppm levels were analyzed by GC-EC after concentration into hexane. A semi-preparative HPLC silica column (25 cm × 9.4 mm i.d., 6 um spheres) provided complete separation of organochlorine compounds and partial separation of organophosphorous pesticides tested from at least 500 mg of fat. This column was shown to be superior to the “official” (AOAC) Florisil column with regard to resolution, speed of elution, solvent volume required, and loading capacity. The column, which operated at moderate pressures of >1000 psi, proved to be resilient and reproducible to repeated sample injections, frac tionations and purgings made from either end.     

预衍生化-气相色谱-质谱法测定水中酸性除草剂

向水样中逐滴加硫酸(1+2)调节至pH<2,用二氯甲烷先后提取3次。合并的提取液用无水酸性硫酸钠脱水,并蒸缩至约1 mL后用吹氮法蒸干。加入丙酮溶解残渣后,在催化剂碳酸钾存在下用五氟苄基溴进行衍生化,溶于正己烷中的衍生产物用极性硅胶柱进行纯化。先用甲苯-正己烷(1+6)混合溶剂淋洗净化柱以除去衍生产物中的干扰副产物,然后用甲苯-正己烷(9+1)从净化柱上将目标化合物洗脱,所得洗脱液经浓缩并加入八氟联苯作内标后供气相色谱-质谱分析。气相色谱中用HP-5MS毛细管色谱柱进行分离,质谱分析中用负化学离子源及选择离子扫描(SIM)。此方法对测定的14种酸性除草剂的检出限(3S/N)均小于10 ng.L-1。对所述14种除草剂做了回收试验,结果在66.0%~117.0%之间,测定7次的相对标准偏差均小于9%。     

Determination of cocaine and norcocaine in plasma and cell cultures using high-performance liquid chromatography

A new simple high-performance liquid chromatographic (HPLC) method was developed for the determination of cocaine and norcocaine. Cocaine and norcocaine in biological samples were buffered to pH 9.0, extracted with diethyl ether and reextracted in a 0.1% aqueous solution of tetramethylammonium hydrogen sulfate (TMAHS) with a theoretical yield of extraction of 100%. The HPLC elution of cocaine and norcocaine was performed using a Spherisorb RP-18, 100 mm x 4.6 mm I.D., 5 microns particle size column with a mobile phase containing acetonitrile-0.1% TMAHS aqueous solution (60:40). The compounds were entirely separated, and a reliable limit of quantitation was set at 20 ng/ml when extracted from 0.5 ml of plasma. No interference with 26 other drugs was found. Cocaine and norcocaine stability studies showed that their half-lives in human plasma incubated at 37 degrees C were 50.8 and 43.2 min, respectively. In contrast, plasma from dogs or rats exhibited only weak or no enzymatic esterase activity towards cocaine and norcocaine resulting in less rapid degradation. Hydrolysis could be efficiently inhibited with sodium fluoride and prevented by storage of the sample at -20 degrees C. The highly sensitive assay also allowed the assessment of the oxidative metabolism pathway of cocaine to norcocaine in primary rat hepatocyte cultures.     

A simple preparation of stationary phase of alkylamide reversed-phase liquid chromatograph for the separation of basic substances.

A simple preparation process of alkylamide phase for reversed-phase HPLC (RP-HPLC) is described. The process includes aminopropyltrimethoxysilane firstly reacted with octanoyl chloride, then the intermediate was coupled onto porous silica. The resultant bonded silica has a reproducible ligand surface concentration and homogenous bonded ligand distribution on the porous silica. Characterization of prepared packing was carried out with elemental analysis, solid-state 13C NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes under methanol/water as binary mobile phase. The results showed that the stationary phase have excellent chromatographic properties and can be efficiently used for the separation of basic compounds.     

Chemical compositional separation of styrene-methyl methacrylate copolymers using high-performance liquid chromatography with liquefied carbon dioxide as eluent

Chromatographic separation of styrene-methyl methacrylate (MMA) copolymers depending on the chemical composition was studied using liquefied carbon dioxide as an adsorption promoting solvent, and tetrahydrofuran, chloroform containing ethanol as a desorption promoting solvent in the mobile phase and the column packed non-bonded silica gel by a solvent gradient method. With the increase of MMA content, the elution was retarded indicating that the typical normal-phase type of adsorption occurred. The effects of type of desorption solvents, molecular mass of sample, and column temperature on the elution were investigated.     

High-performance liquid chromatographic determination of phenolic compounds in rice

A method has been developed for the determination of 6'-O-feruloylsucrose, 6'-O-sinapoylsucrose, ferulic acid, sinapinic acid, p-coumaric acid, chlorogenic (3-caffeoylquinic) acid, caffeic acid, protocatechuic acid, hydroxybenzoic acid, vanillic acid, and syringic acid in rice. The rice samples were extracted with 70% ethanol, filtered, and defatted. The defatted aqueous solution was subjected to solid-phase extraction using a C18 silica gel cartridge; no analyte was lost in this procedure. The 70% acidic methanol elution was analyzed directly by HPLC and HPLC-ESI-MS. Phenolic compounds were separated with a C18 reversed-phase column by gradient elution using 0.025% trifluoroacetic acid in purified water (A)--acetonitrile (B) (0 min, 5% B; 5 min, 9% B; 15 min, 9% B; 22 min, 11% B; and 38 min, 18% B) as the mobile phase at a flow rate of 0.8 ml/min. Detection limits ranged from 0.10 to 0.35 ng per injection (5 microl). Relative standard deviations of 0.22-3.95% and recoveries of 99-108% were obtained for simultaneous determination of these phenolic compounds. This method was applied to analysis of phenolic compounds in brown rice and germinated brown rice soaked in 32 degrees C water for varying durations.     

High-performance liquid chromatographic determination of mitoxantrone in liposome preparations using solid-phase extraction and its application in stability studies

A method for the determination of the entrapped mitoxantrone in liposome preparations was developed. The method consists of a solid-phase extraction procedure followed by HPLC analysis. A C₁₈ cartridge was used for solid-phase extraction and 0.5M methanolic HCl was used for elution. The extraction demonstrated a good separation of the mitoxantrone from the phospholipid. A C₁₈ column and a mobile phase containing acetonitrile-0.01 M monopotassium phosphate (40:60) with the pH adjusted to 3.0 with orthophosphoric acid were employed. The detection wavelength was 242 nm. The HPLC method was stability indicating and was applied to determine the degradation of the entrapped mitoxantrone in liposomes. A pseudo-first-order reaction was found for the degradation of the entrapped mitoxantrone. The half-life of the mitoxantrone decreased with increasing pH of the medium. The results demonstrate that the proposed method is satisfactory for the determination of the stability of mitoxantrone in liposome preparations.     

Coupling of a Modified In-Tube Solid Phase Microextraction Technique with High Perfor- mance Liquid Chromatography-Fluorescence Detection for the Ultra-Trace Determination of Polycyclic Aromatic Hydrocarbons in Water Samples

A modified in-tube solid phase microextraction (SPME) technique in conjunction with a high performance liquid chromatography (HPLC) was developed for the trace determination of polycyclic aromatic hydrocarbons (PAHs) in water samples. The extraction device contained a regular HPLC syringe, replacing the metallic needle by two concentric fused silica capillary tubes. The capillary tubes were coated with polydimethylsiloxane (PDMS) as the sorbent and were attached to the syringe by a homemade interface made from polyether ether ketone (PEEK). The sorption of analytes was achieved by frequent withdrawing and ejecting the water sample from/into a vial via the capillary tubes. For the desorption step, an aliquot of organic solvent was withdrawn and subsequently injected directly into the HPLC system. Limits of detection for the elected PAHs were between 0.001 and 0.006 g L^–1 with a RSD of 2.6–6.3%.     

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.     

A rapid HPIC method for the quantitative determination of tetramethylammonium chloride in some aqueous solutions used as food simulants

Summary The present paper reports a rapid HPIC method for the determination of tetramethylammonium chloride in aqueous matrices which are used as food simulants in migration tests. These simulants were: bidistilled water, an aqueous solution of 3% acetic acid, an aqueous solution of 15% ethyl alcohol, a mixed aqueous solution of 3% acetic acid and 15% ethyl alcohol. The method, as developed, has been applied to the determination of unreacted tetramethylammonium chloride residues which are able to migrate from finished epoxy resins which are used as food packaging materials.Research partially supported by National Research Council of Italy, Special Project RAISA, sub-project 4, Paper N^o 695.     

Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.     

The high pressure liquid chromatographic separation of cis and trans dienone-iron carbonyl complexes and of ψ-endo and ψ-exo dienol-iron carbonyl complexes on 10 m silica

High Pressure Liquid Chormatography (HPLC) has been used to separate cis and trans dienone-iron carbonyl complexes having different σ and X bonded moieties, and ψ-endo and ψ-exo dienol-iron carbonyl complexes. TLC predicts the appropriate solvent polarity needed and elution order on microparticulate silica columns. Data is reported for methylene chloride and methylene chloride-acetonitrile mobile phases on 10 μm silica.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

Determination of anti‐malaria agent chloroquine using single drop liquid‐liquid‐liquid microextraction

A simple, sensitive, and inexpensive single drop liquid‐liquid‐liquid microextraction combined with isocratic RP‐HPLC and UV detection was developed for the determination of anti‐malaria drug, chloroquine. The target compound was extracted from alkaline aqueous sample solution (adjusted to 0.5 mol/L sodium hydroxide) through a thin layer of organic solvent membrane and back‐extracted to an acidic acceptor drop (adjusted to 0.02 mol/L phosphoric acid) suspended on the tip of a 25 μL HPLC syringe in the organic layer. This syringe was also used for direct injection after extraction. The linear range was 1–200 μg/L. The LOD and LOQ were 0.3 and 1.0 μg/L, respectively. Intra‐and inter‐day precisions were less than 2.0 and 2.3%, respectively. The real samples were successfully analyzed using the proposed method. The recoveries of spiked samples were more than 94.6%.     

高效液相色谱-电感耦合等离子体质谱法同时测定果蔬中6种砷形态

建立了一种同时测定果蔬中亚砷酸根、砷酸根、砷胆碱、砷甜菜碱、一甲基砷酸和二甲基砷酸等6种砷形态的高效液相色谱-电感耦合等离子体质谱分析方法。样品经甲醇水提取,采用阴离子分析柱,50 mmol/L碳酸铵溶液和水作为流动相进行梯度洗脱,高效液相色谱分离,电感耦合等离子体质谱进行定性和定量分析。在0.5～50μg/kg范围内...     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Determination of captopril in human plasma with precolumn derivatization using solid phase extraction and HPLC

A rapid, accurate and sensitive method for the determination of captopril in human plasma was developed by solid phase extraction and high performance liquid chromatography (HPLC), using precolumn derivatization of captopril with chromophore label o-phethaldialdehyde (OPA). The extraction of captopril from human plasma was carried out by an amino propyl cartridge. A 0.01 M solution of HCl in methanol showed the best recovery and was chosen for elution of captopril in cartridge. This methanolic solution was applied to react with aqueous solution of OPA and glycine as a coderivatization reagent. The process of derivatization was completed within 2 min at room temperature. The derivatized captopril was injected into a reverse phase HPLC system. Mobile phase was consisted of water:acetonitrile:trifluoroacetic acid (85:15:0.1 v/v/v) with a flow rate of 1 ml min^?1 and detector was used at 345 nm. Linear dynamic range and limit of detection were found as 0.1–6 ppm and 0.1 ppm, respectively.