Analysis of vinca alkaloids in plasma and urine using high-performance liquid chromatography with electrochemical detection

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.     

Determination of vinca alkaloids in plasma and urine using ion-exchange chromatography on silica gel and fluorescence detection

The development of a method for the determination of the antineoplastic vinca alkaloids vinblastine and vindesine in biological samples is described. The selectivity of the assay is high owing to the use of solid-phase extraction on a cyanopropyl extraction column prior to isocratic chromatography on unmodified silica gel with fluorescence detection. The influence of acetonitrile concentration and mobile phase pH on the capacity factors of the drugs was studied in order to optimize the separation between the drugs and endogenous components. The effect of varying the type and concentration of competing cations in the mobile phase was also examined. The limit of determination (signal-to-noise ratio = 3) for vinblastine is 0.5 ng/ml in plasma and urine and for vindesine 2.5 ng/ml. The assay is suitable for determining the concentrations of both compounds in plasma and urine samples from patients.     

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine.

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C18 solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5.10(-4) M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.     

An HPLC method for determination of azadirachtin residues in bovine muscle

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.     

新型纳米纤维固相萃取柱制备及其用于组织样品中柔红霉素测定研究

研制出一种新型纳米纤维固相萃取柱,并将其用于大鼠心、肝、脾、肺、肾组织及血浆中柔红霉素的净化、浓缩处理,建立了生物样品中柔红霉素的高效液相荧光检测法.以10%(V/V)HClO4为溶剂,对组织样进行匀浆和离心处理,上清液用纳米纤维小柱净化富集,以50 μL含1%(V/V)冰醋酸的甲醇洗脱,进样20μL检测.流动相为甲醇...     

High-performance liquid chromatographic determination of vinca-alkaloids in plasma and urine

A liquid chromatographic method is described for separating and determining vinblastine, vincristine and vindesine in plasma and urine. The drugs are extracted from the biological material using an ion-pair extraction, with sodium octylsulphate as counter-ion at pH 3. The extracts are injected on a reversed-phase system with a cyano column as stationary phase and a mobile phase composed of acetonitrile-phosphate buffer, pH 3 (65:35, vol. %). Stability studies are carried out for stock solutions of the drugs in water at different temperatures and pH values. The stability of these compounds in plasma is also investigated in the presence of an antioxidant. The method is applied to determine drug levels of vindesine and vinblastine in preliminary pharmacokinetic studies, using vincristine as the internal standard.     

High-performance liquid chromatographic determination of imidocarb in cattle kidney with cation-exchange clean-up.

A high-performance liquid chromatographic (HPLC) method for the determination of the antiprotozoal agent imidocarb in cattle kidney is developed. The drug is extracted from tissue with acetone in the presence of base. The extract is partitioned between saturated salt and chloroform and the organic layer evaporated to dryness. Clean-up is by cation-exchange solid-phase extraction on a carboxylic acid column. HPLC analysis is carried out on a Spherisorb S3W-PC18 column with ultraviolet detection at 260 nm. Average recoveries at the 0.05 and 0.01 mg kg-1 levels are 77.5 and 76.3%, respectively. The limit of detection is 0.001 mg kg-1.     

Rapid and sensitive analysis of vincristine in human plasma using on-line extraction combined with liquid chromatography/tandem mass spectrometry

A simple and rapid method has been developed and validated for the quantitation of vincristine in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) with atmospheric pressure chemical ionization using on-line solid-phase extraction. The method uses vinblastine as internal standard and the sample preparation is limited just to a plasma protein precipitation step. Further sample clean-up is carried out on-line through a perfusion column preceding an analytical phenyl LC column, the latter directly connected to the mass spectrometer. Quantitation is performed in multiple reaction monitoring mode using the transitions of m/z 825.3 --> 765.3 and 811.3 --> 751.3 for vincristine and vinblastine respectively. The assay was linear (r2 > or =0.99) in a concentration range from 0.1 to 500 ng/mL. Carry-over, measured on the experimental set-up, was less than 0.04%. Recovery for vincristine and the internal standard was within 90-95%. The intra-day and inter-day assay precision ranged from 1.2% to 6.8% RSD while mean percentage deviation from nominal value ranged from 0.01% to 6.1%. The proposed assay was found suitable for pharmacokinetics investigations and clinical therapeutic drug monitoring especially in pediatric cancer patients.     

Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments

A method is described for the qualitative and semi-quantitative simultaneous determination of both non-macrocyclic and macrocyclic trichothecene biotoxins from samples derived from indoor environments. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation followed by tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyser. Aqueous methanol was used in the initial extraction and solvent partitioning and solid-phase extraction in the purification of samples. The HPLC separation was run on-line with electrospray ionization MS-MS detection. The detection limits and recoveries of the procedure varied from 1 to 1000 pg and from 31 to 92%, respectively. As the method includes few and not very labour intensive sample treatment steps, it should allow for a high throughput of samples with good prospects of automation.     

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.     

High-performance liquid chromatographic method for the simultaneous determination of the enantiomers of carvedilol and its O-desmethyl metabolite in human plasma after chiral derivatization

Quantitative methodology for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of carvedilol, a new multiple-action antihypertensive agent exhibiting both vasodilator and beta-blocking activity, and its active metabolite, O-desmethylcarvedilol, in human plasma is described. The method involves reversed-phase solid-phase extraction of the analytes, followed by derivatization of the extract with the chiral reagent, 2,3,4,6,-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate and injection of the resultant diastereoisomers onto a reversed-phase HPLC column coupled to a fluorescence detector. Both pairs of diastereoisomers formed are completely resolved within 12 min (resolution for the respective pairs is 2.26 and 3.32) and the baseline is clean and free from extraneous peaks. The assay is linear over the range 0.6-80 ng/ml of human plasma with a lower limit of detection of approximately 100 pg on-column for each of the enantiomers. The method can be adapted for a number of structural analogues of carvedilol and is currently applied in support of preclinical and clinical studies of the drug.     

Chromatographic determination of vitamins in foods.

Chromatographic methods for the determination of water- and fat-soluble vitamins in foods are reviewed. For each vitamin, sample preparation, detection problems and chromatographic conditions are presented and discussed. High-performance liquid chromatography (HPLC) is becoming a standard method in vitamin assay, especially for routine work. HPLC systems can be automated using in-line solid-phase extraction and column switchings, resulting in very sensitive methods, even when simple UV detection is employed.     

Determination of some aldehydes by using solid-phase microextraction and high-performance liquid chromatography with UV detection

A new approach has been developed for the extraction and determination of aldehydes such as veratraldehyde, m-nitrobenzaldehyde, cinnamaldehyde, benzaldehyde, and p-chlorobenzaldehyde by using solid-phase microextraction (SPME) and high-performance liquid chromatography with UV detection (HPLC/UV). The method involves adsorption of the aldehydes on polydimethylsiloxane/divinylbenzene-coated fiber, followed by desorption in the desorption chamber of the SPME-HPLC interface, using acetonitrile-water (70 + 30) as the mobile phase; UV detection was at 254 nm. A good separation of 5 aldehydes was obtained on a C18 column. The detection limits of veratraldehyde, m-nitrobenzaldehyde, cinnamaldehyde, benzaldehyde, and p-chlorobenzaldehyde are 25, 41, 13, 12, and 11 pg/mL, respectively, which are about 100 times better than the detection limits for other SPME methods using gas chromatography. The proposed method was validated by determining benzaldehyde in bitter almonds and cinnamaldehyde in cinnamon bark. The recoveries of the 5 analytes were determined by analysis of spiked drinking water.     

Determination of cephalosporins in raw bovine milk by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction was developed for the determination of cefazolin, cefoperazone, cefquinome and ceftiofur in raw bovine milk. The milk fat was removed by centrifugation and the cephalosporins were extracted in acetonitrile. The extract was cleaned up by solid-phase extraction on an octadecyl sorbent. The compounds were separated by ion-paired gradient HPLC on a phenyl column with ultraviolet detection at 270 nm. The limits of detection estimated by a conservative model were 11 microg/kg for cefazolin and cefoperazone and 7 microg/kg for cequinome and ceftiofur. The mean recoveries were 86-88% for cefazolin, 91-93% for cefoperazone, 69-72% for cefquinome and 84-88% for ceftiofur in the concentration range 20-200 microg/kg.     

Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography

Two direct HPLC analytical methods for the screening of the major indole alkaloids of Catharanthus roseus hairy roots and their iridoid precursors have been developed. Photodiode array and fluorescence detection were performed. The separation was achieved on a reversed-phase C18 column. The first method allowed the separation of catharanthine, serpentine, tabersonine, vindoline, vinblastine, and vincristine in 20 min. Ajmalicine, tryptophan, tryptamine and secologanine were separated using the second method in 13 min. The identification of the compounds was based on the retention time and the comparison of UV spectra with those of authentic standards. A simplified alkaloid extraction method was developed in order to accelerate sample preparation. The assays were successfully used to quantify major compounds of the secondary metabolism of hairy root cultures of C. roseus, thus providing a reliable tool for rapid screening of C. roseus secondary metabolite samples. In these cultures, ajmalicine, serpentine, catharanthine, tabersonine, and tryptamine were detected, but tryptophan, vindoline, vinblastine and vincristine were not.     

Multiwalled carbon nanotubes as sorbent for on-line coupling of solid-phase extraction to high-performance liquid chromatography for simultaneous determination of 10 sulfonamides in eggs and pork

The sulfonamides (SAs) have been widely used as effective chemotherapeutics and growth promoters in animals' feeding, but their residues could be a potential danger to human health due to their carcinogenic potency and possible antibiotic resistance. Development of a simple and sensitive method for the determination of SAs residues in food of animal origin, therefore, is of great significance. An on-line solid-phase extraction (SPE) method using multiwalled carbon nanotubes as sorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous determination of 10 sulfonamides (SAs) in eggs and pork was developed. The adsorptive potential of carbon nanotubes for solid-phase extraction of sulfonamides was investigated for the first time in the present paper. To on-line interface solid-phase extraction with HPLC, a conventional sample loop on the six-port injector valve of the HPLC was replaced by a preconcentration column packed with carbon nanotubes. The analytes in water solution were preconcentrated onto the preconcentration column and subsequently eluted with mobile phase of methanol-water (22:78). The developed on-line solid-phase extraction method for HPLC permitted the current HPLC separation and the next preconcentration proceeded in parallel, and thus allows one determination finished within 35 min. The RSD of 10 SAs for nine replicate measurements of a standard mixture of 1 microgl(-1) were in the range of 2.5-7.8%. The method was applied to the determination of trace sulfadiazin (SDZ), sulfamerazine (SMR), sulfadimidine (SDMD), sulfathiazole (STZ), sulfamoxol (SMO), sulfamethizole (SMT), sulfamethoxypyridazine (SMP), sulfachlorpyridazine (SCP), sulfadoxin (SDX) and sulfisoxazole (SIA) in eggs and pork. The results indicated that the proposed method was simple, cost-effective and sensitive.     

A sensitive liquid chromatography–tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: Application to pharmacokinetic study

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 μl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 μm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5–2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.     

Determination of forchlorfenuron residues in watermelon by solid-phase extraction and high-performance liquid chromatography

A method using solid-phase extraction for cleanup, followed by high-performance liquid chromatography with ultraviolet detection (HPLC/UV), was developed for the determination of forchlorfenuron residues in watermelon. The pesticide is extracted from the sample with acidic acetonitrile, and the extract is loaded onto a primary-secondary amine (PSA) column. The pesticide is eluted with acetone and determined by HPLC/UV. The PSA column was found to provide effective cleanup, removing the greatest number of sample matrix interferences. The acetonitrile extraction followed by the PSA cleanup provided recoveries of >95%, coefficients of variation (precision) of <10%, and sensitivity of 0.005 mg/kg, in agreement with the directives for method validation in residue analysis. The proposed method was successfully used to determine forchlorfenuron residue levels and dissipation rates in watermelon grown in an experimental greenhouse in Beijing, People's Republic of China.     

SPE-RP-HPLC法测定羊肉组织中胺菊酯和三氟氯氰菊酯残留量

建立了羊肉组织中胺菊酯和三氟氯氰菊酯的固相萃取-反相高效液相色谱测定法。采用氟罗里硅土固相萃取柱(1000 mg/6 mL)进行固相萃取。以Shim-pack VP-ODS(200 mm×4.6 mm)柱为分析柱,流动相为甲醇:水=95:5(V/V),流速为0.7 mL/min。胺菊酯和三氟氯氰菊酯分别在0.01～6.40μg/mL(r=0.9999)和0.068～7.20ug/mL(r=0.9998)范围内与峰面积呈良好线性关系,检出限分别为0.001μg/mL和0.002μg/mL,胺菊酯和三氟氯氰菊酯的回收率为90.2%～101.4%,相对标准偏差为2.3%～4.0%。该方法可作为羊肉组织中胺菊酯和三氟氯氰菊酯含量监测的控制方法。     

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.