Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS)

Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol-acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm x 2.1 mm ID, particle size 5 microm) with 1 mM ammonium hydroxide in water-acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 microL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50-5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide.     

Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography–mass spectrometry

A sensitive HPLC–MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C₁₈ cartridges followed by HPLC–MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.     

Simultaneous determination of cyclophosphamide and carboxyethylphosphoramide mustard in human plasma using online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 μm C18 110A, 100 mm × 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between ?6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.     

Direct gas chromatographic determination of dechloroethylcyclophosphamide following microsomal incubation of cyclophosphamide

A method for the sensitive determination of dechloroethylcyclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C₈ cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.     

Simultaneous analysis of cyclophosphamide, doxorubicin and doxorubicinol by liquid chromatography coupled to tandem mass spectrometry

A method for the simultaneous determination of cyclophosphamide (CP), doxorubicin (dox), and doxorubicinol (dol) was developed and validated to analyze 400 microL of plasma from patients receiving chemotherapeutic treatment with CP and dox. Final calibration ranges for the analytes were 0.440-60.0 microg/mL for cyclophosphamide, 7.20-984 ng/mL for dox and 3.04-104 ng/mL for dol. The samples were prepared using solid phase extraction and analyzed using a gradient separation over a Waters Symmetry C18, 2.1 by 30 mm (Milford, MA) column. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer.     

High-performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT-11, and its metabolites SN-38 and SN-38 glucuronide in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT

We established a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the camptothecin (CPT) derivative, irinotecan hydrochloride (CPT-11) and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT. Plasma samples were pretreated with 0.146 M H₃PO₄ to inactivate carboxylesterase and β-glucuronidase in rat plasma, and added with the internal standard solution (0.146 M H₃PO₄ containing 1 μg/ml CPT) and then analyzed. The method was validated for CPT-11 (5 to 25 000 ng/ml), SN-38 (5 to 2500 ng/ml) and SN-38G (2.5 to 500 ng/ml). This method enabled the determination of many samples within a relatively short time with easy sample preparation. It also had four advantages compared with conventional determination methods, i.e. automation of a complicated sample preparation, time-saving by the simultaneous determination of three compounds, the direct determination of SN-38G, and the small amount of plasma required for the determination.     

A rapid microtechnique for the preparation of biological material for the simultaneous analysis of calcium,magnesium and protein

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r²) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.     

Analysis of polysaccharide-poly(ethylene-co-acrylic acid) composites by fourier-transform infrared spectroscopy

A method, based on absorbances in FTIR spectra of the C---O singlebond stretch band of the polysaccharide and the C---H stretch band of poly(ethylene-co-acrylic acid) (EAA) at 2851 cm⁻¹, was developed for the determination of EAA in composites with either dextran or starch. Spectral subtraction of the polysaccharide component was necessary for quantitative determination of EAA. The accuracy of this analytical procedure is affected by the fact that absorbances of these two bands are not equally dependent on particle size of the sample in the KBr pellet; the absorbance ratio, as calculated from FTIR spectra therefore varies with sample preparation conditions. To ensure the necessary particle size uniformity during the preparation of KBr pellets, a method based on relative intensities of two bands in the C---O region of the spectrum was developed as an indicator of particle size in the pellet. This method was also used for monitoring sample size in the polysaccharide standard used for spectral subtraction, since particle size uniformity between sample and polysaccharide standard was also necessary for accurate determination of EAA.     

Determination of the concentration of ligand-specific molecules, found in mixtures with ligand-nonspecific molecules

A new method, which allows determination of the concentration of ligand-specific molecules in a mixture of these molecules with biochemically similar but ligand-unspecific molecules, is suggested. The method is based on a partial exhaustion of the mixture on a column with immobilized ligand and determination of the part of ligand-specific molecules presented in exhausted mixture. The concentration of monoclonal antibodies specific to bovine serum albumin in a commercial "Sigma" preparation and concentration of polyreactive immunoglobulins in a commercial "Sigma" preparation of bovine immunoglobulins were determined by suggested method.     

Stable and sensitive method for the simultaneous determination of N-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxymethotrexate in biological fluids

A high-performance liquid chromatographic method for the determination of N⁵-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.     

The effect of plasma on the reaction of cyclophosphamide with 4(-p-nitrobenzyl)-pyridine.

Cyclophosphamide is not structurally modified by blood plasma and may be recovered quantitatively from it. The apparent loss of alkylating ability of cyclophosphamide, as measured by its ability to react with 4-(p-nitrobenzyl)-pyridine (NBP), following treatement with plasma is not, as has been reported, due to metabolism of the drug but rather to an inhibition of the colorimetric reaction by a constituent of the plasma. This inhibition is strongly pH dependent, reaching 100% when the pH of the solution of cyclophosphamide in plasma is above 8, and falling to less than 20% when the pH is below 6, but extraction with chloroform at pH 7 separates cyclophosphamide from the inhibitor. Although the nature of the inhibitor has not been elucidated, its presence in plasma is of great importance in the quantitative determination of cyclophosphamide, and may also be of significance in the biological effects of cyclophosphamide and other alkylating agents in vivo.     

Determination of levomycetin in the aerosol preparation, "Levovinysol"]

A spectrophotometric method for determination of levomycetin in "Levovinysol", an antiburn aerosol preparation was developed. The determination was performed after levomycetin extraction with a hydrochloric acid solution from the composition. The results of the determination were statistically treated.     

High-performance liquid chromatographic determination of the enantiomers of cyclophosphamide in serum

A high-performance liquid chromatographic (HPLC) achiral-chiral coupled assay to measure the serum concentration of the enantiomers of cyclophosphamide is described. The R- and S-enantiomers of cyclophosphamide were quantified using a 5-cm-long C₁ Spherisorb 5-μm column, with switching of the eluent containing racemic cyclophosphamide onto a 10-cm-long α₁, acid glycoprotein column. The limit of determination was 1.25 mg l⁻¹ for each enantiomer and the ratio of the enantiomers over the range 2.5 to 100 mg l⁻¹ was 1. Serum enantiomer concentrations in blood samples taken from patients receiving 0.30 to 0.75 g m⁻² of intravenous racemic cyclophosphamide could be measured at least three half-lives post dose. In six patients no significant difference in the clearance of R- and S-cyclophosphamide was found.     

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC₅₀ values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.     

Determination of cyclophosphamide metabolites by gas chromatography and thermionic specific detection interindividual differences in hepatic biotransformation of cyclophosphamide in man in vitro

Sensitive methods for the determination of the cyclophosphamide metabolites nornitrogen mustard, 4-ketocyclophosphamide and carboxyphosphamide are presented. After liquid-liquid extraction and derivatization, the metabolites are determined by gas chromatography and thermionic specific detection. The methods were used to study the in vitro biotransformation of cyclophosphamide with S-9 liver fractions of human donors. The results show large interindividual differences in the formation of nornitrogen mustard and carboxyphosphamide. 4-Ketocyclophosphamide was not detected.     

A new method for differential determination of basic natural forms of indolyl-3-acetic acid

A new ELISA method is proposed for differential quantitative determination of free (indolyl-3-acetic acid; IAA) and bound (indolyl-3-acetyl-L-aspartate) forms of natural auxins. There is similarity in results obtained by this and some traditionally used methods. The standard error of determination of the active form of IAA by our method is 1.5-2.0 times less than that using the traditional method. The method of quantitative differential determination of the main natural auxins does not require preliminary sample preparation, and this shortens assay time. The developed method has been used for practical determination of different forms of endogenous IAA in wheat and dandelion ovaries subjected to minimal treatment. This method can be used to investigate changes in the ratio of various hormonal forms of auxins that differ in their physiological activity in reproductive organs of angiosperms at various stages of reproduction.     

Trimethyllead acetate: a first-choice heavy atom derivative for protein crystallography

The three-dimensional conformation of a protein provides a wealth of biochemical information and with the advent of cloning techniques that allow the preparation of proteins almost at will, a renewed interest has arisen in the crystallographic determination of protein structures. As in any research technique, however, there are often many difficulties encountered in an X-ray crystallographic investigation. One of these is the "phase problem." Although in recent years there has been considerable progress in the development of techniques for phase determination, including the use of molecular replacement and multiple wavelength measurements, the multiple isomorphous replacement method is still the most successful method for obtaining a three-dimensional structure. Here we report the use of trimethyllead acetate as a heavy atom compound of first choice in the preparation of an isomorphous heavy atom derivative.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.     

Rapid method for the determination of piroxicam in rat plasma using high-performance liquid chromatography

A previously published method was used for the determination of piroxicam in plasma samples obtained from rat. The sample preparation involved liquid extraction, centrifugation and evaporation. Separation of piroxicam from internal standard occurred on a reversed-phase C₁₈ column with a mobile phase consisting of methanol-phosphate buffer pH 2 (45:55). The detection limit of the assay was 0.02–20 μg/ml. The assay linearity was good (typically r = 0.9992). The method was applied for determination of piroxicam in rats after administration of an oral dose of 2 mg/kg piroxicam.