Determination Of Cyclophosphamide Research Articles

Application of Charge Transfer Reactions for the Quantitative Spectrophotometric Determination of Cyclophosphamide in Pure and Pharmaceutical Formulation

Abstract: The present investigation describes two simple, sensitive and validated spectrophotometric methods for the determination of Cyclophosphamide (CP) in pure and formulated forms. The methods are mainly based on the formation of charge transfer (CT) complex, with the reagents 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (Method A) and p-Chloranilic acid (PCA) (Method B), hence a sta-ble, 1:1 stoichiometric complex was obtained, which shows orangeyellow and pink colors, respectively. The produced chromogens optical densities were measured at 352 and 522 nm, respectively. The Beer’s law was tested in the range of 2-16 and 5-50 µg mL -1 . The optical characteristics such as molar absorptivity (ɛ), and San-dell’s sensitivity values were calculated. Other analytical parameters like, Limit of Detection (LOD), Limit of Quantifica-tion (LOQ), accuracy and precisions were evaluated. The proposed methods were successfully applied for the determina-tion of the CP in tablet formulation with higher accuracy, better recoveries and less than 2% relative standard deviation.

Antineoplastic drugs contamination of workplace surfaces in two Portuguese hospitals.

Despite the classification as known or suspected human carcinogens, by the International Agency for Research on Cancer, the antineoplastic drugs are extensively used in cancer treatment due to their specificity and efficacy. As human carcinogens, these drugs represent a serious threat to the healthcare workers involved in their preparation and administration. This work aims to contribute to better characterize the occupational exposure of healthcare professionals to antineoplastic drugs, by assessing workplace surfaces contamination of pharmacy and administration units of two Portuguese hospitals. Surface contamination was assessed by the determination of cyclophosphamide, 5-fluorouracil, and paclitaxel. These three drugs were used as surrogate markers for surfaces contamination by cytotoxic drugs. Wipe samples were taken and analyzed by HPLC-DAD. From the total of 327 analyzed samples, in 121 (37%) was possible to detect and quantify at least one drug. Additionally, 28 samples (8.6%) indicate contamination by more than one antineoplastic drug, mainly in the administration unit, in both hospitals. Considering the findings in both hospitals, specific measures should be taken, particularly those related with the promotion of good practices and safety procedures and also routine monitoring of surfaces contamination in order to guarantee the appliance of safety measures.

English

A new, simple, rapid, wide applicable range and reliable second derivative spectrophotometric method has been developed for determination of cyclophosphamide (CP) in bulk and pharmaceutical dosage forms. Calibration graph is linear in the concentration range of 25 - 200 μg/ml of CP with 10 μg/ml of detection limit and correlation coefficient of 0.9976. The precision and accuracy were acceptable depending upon the values of relative standard deviation and error percentage. Developed second derivative spectrophotometric method can be directly and easily applied for analyzing pure form and commercial pharmaceutical preparations of CP. The method was compared with a standard high performance liquid chromatography (HPLC) method and can be used for the quality control of pharmaceutical preparations in Erbil City. Key words : Cyclophosphamide, derivative spectrophotometry.

Monolithic packed 96‐tips set for high‐throughput sample preparation: determination of cyclophosphamide and busulfan in whole blood samples by monolithic packed 96‐tips and LC‐MS

A monolithic methacrylate packed 96-tips device was used for the extraction of the busulfan and cyclophosphamide in whole blood samples. Using a packed 96-tips set, a 96-well plate could be handled in about 2 min. The key aspect of the monolithic phase is that monolithic material can offer both good extraction capacity and low-back-pressure properties. The validation of the methodology showed that the accuracy values of quality-control samples were between 99 and 113% for busulfan, and between 103 and 110% for cyclophosphamide. The inter-day precision ranged from 7.0 to 12% for busulfan and from 13 to 16% for cyclophosphamide. The standard calibration curves were obtained within the concentration range 5-2000 nm for busulfan and from 10 to 5000 nm for cyclophosphamide in blood samples. The coefficients of determination were ≥0.99.

Quantitative determination of cyclophosphamide in rat plasma using an on-line SPE HPLC-DAD

Quantitative determination of cyclophosphamide in rat plasma using an on-line SPE HPLC-DAD

Determination of cyclophosphamide and ifosfamide in sewage effluent by stable isotope-dilution liquid chromatography–tandem mass spectrometry

A reliable and specific method was developed for the determination of the cytotoxic drugs cyclophosphamide and ifosfamide in sewage effluent. The most successful combination was found to be Strata-X solid-phase extraction followed by Florisil ® clean-up with analysis by liquid chromatography–tandem mass spectrometry. Quantification by internal standardisation was achieved using custom synthesised d4-cyclophophosphamide. The mass spectrometer was operated in highly selective reaction monitoring (HSRM) mode, which significantly reduced matrix noise and improved sensitivity. Although it suffered from some ionisation suppression, electrospray ionisation (ESI) was found to give an order of magnitude better sensitivity in terms of limit of detection than atmospheric pressure chemical ionisation (APCI). Using final effluent from two different sewage treatment plants, the method was validated following official European guidelines and shown to be a high performance tool for routine analysis at the sub-nanogram per litre level. Depending on the matrix, the limit of detection for cyclophosphamide was between 0.03 ng/L and 0.12 ng/L and for ifosfamide between 0.05 ng/L and 0.09 ng/L. For cyclophosphamide the accuracy and precision, tested at 1.7 ng/L, were 98–109% and ≤13%, CV respectively. For ifosfamide the accuracy and precision, tested at 1.1 ng/L, were 98–113% and ≤15% CV, respectively. Depending on the sample matrix the absolute recovery of the internal standard was between 57% and 70%. The method was tested by analysis of spot samples taken from the final effluent discharges of two sewage treatment plants; the first using a conventional trickling filter treatment process and second employing activated sludge followed by ultra violet treatment. Cyclophosphamide was detected at 0.19 ng/L at the first plant and at the second detected at 3.7 ng/L and 3.5 ng/L, before and after the UV treatment process; ifosfamide was not detectable at either plant.

Rapid and Sensitive Method for Determination of Cyclophosphamide in Patients Plasma Samples Utilizing Microextraction by Packed Sorbent Online with Liquid Chromatography‐Tandem Mass Spectrometry (MEPS‐LC‐MS/MS)

The aim of the present investigation was to develop a simple and fast method for the determination of cyclophosphamide in human plasma samples. Microextraction in packed syringe (MEPS) was used as an online rapid sample preparation method, followed by liquid chromatography with tandem mass spectrometry (LC‐MS‐MS) for the quantification of cyclophosphamide. The new method reduced the sample handling and the analysis time by several folds compared to liquid chromatography and UV detection. The limit of detection (LOD) was 0.005 µg/mL and the lower limit of quantification was 0.5 µg/mL. The accuracy of the quality control (QC) samples ranged from 95 to 106%. The inter‐day variation was within the range 5–9% while the intra day variation was between 1–5%. The calibration curve in plasma was constructed within the concentration range 0.5–150 µg/mL. The regression correlation coefficient (r) was ≥0.99 for all runs. The limit of detection improved by 100 time using MEPS‐LC‐MS/MS (0.005 µg/mL) compared to LLE‐LC‐UV (0.5 µg/mL). The present method was employed for the analysis of human plasma samples for more 170 patient samples. The concentrations obtained from LC‐MS‐MS were in good agreement with these obtained from LC‐UV with the ratio of 1.02±0.11. The present method is rapid, reliable, and robust and may be used for therapeutic drug monitoring of cyclophosphamide.

Determination of cyclophosphamide and ifosphamide in urine at trace levels by gas chromatography/tandem mass spectrometry.

A specific and sensitive method based on gas chromatography/tandem mass spectrometry with on-column injection was developed to quantify simultaneously cyclophosphamide and ifosphamide in urine by using trophosphamide as an internal standard. The urine samples were extracted with diethyl ether and derivatization was performed with heptafluorobutyric anhydride. The detection limits of cyclophosphamide and ifosphamide in urine samples were 0.1 and 0.5 ng ml(-1), respectively, with a signal-to-noise ratio of 3 : 1. The sensitivity, the specificity and the low cost of the instrumentation involved make this method suitable for economical analysis on a large scale, such as for the biological monitoring of occupational exposure to cyclophosphamide and ifosphamide in production plants and in hospitals during their pharmacological use.

Determination of cyclophosphamide and ifosphamide in urine at trace levels by gas chromatography/tandem mass spectrometry

Determination of cyclophosphamide and ifosphamide in urine at trace levels by gas chromatography/tandem mass spectrometry

Drugs hazardous to healthcare workers. Evaluation of methods for monitoring occupational exposure to cytostatic drugs.

We review the literature concerning possible health risks for individuals (e.g. healthcare workers and pharmaceutical plant employees) occupationally exposed to cytostatic drugs. Cytostatic drugs possess toxic properties and may therefore cause mutagenic, carcinogenic and teratogenic effects. Hence, individuals handling these drugs in the course of their employment may face health risks. For this reason, it is important to monitor occupational exposure to these drugs. An overview of exposure monitoring methods is presented and their value is discussed. Most studies involve nonselective methods for biological monitoring and biological effect monitoring, such as the urinary mutagenicity assay and analysis of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. The disadvantages of these biological methods are that their sensitivity is low and it cannot be proved beyond any doubt that the results found were caused by occupational exposure to cytostatic drugs. For occupational health services it is important to have sensitive and specific methods for monitoring exposure to cytostatic drugs. One of the most promising methods seems to be the determination of cyclophosphamide in urine using gas chromatography-tandem mass spectrometry. Several studies have demonstrated exposure to cyclophosphamide and other cytostatic drugs, even when protective measures were taken and safety guidelines were followed. To estimate the magnitude of any health effects arising from this exposure, we calculated the risk of cancer due to occupational exposure to cyclophosphamide on the basis of available human and animal dose-response data and the amounts of cyclophosphamide found in urine. The initial results show an extra cancer risk for pharmacy technicians and nurses.

Spectrophotometric and Spectrofluorimetric Determination of Cyclophosphamide and Its Isomer Ifosphamide

Based on the condensation reaction between amino groups of either cyclophosphamide or ifosphamide with the carbonyl group of ninhydrin, giving coloured and fluorescent products, a spectrophotometric procedure for both drugs is suggested in the range from 30 to 90μg ml−1 at 564 nm, with accuracies of 100.6 ± 1.06% and 100.8 ± 1.02% for cyclophosphamide and ifosphamide, respectively. Also, a spectrophotometric procedure for both drugs is proposed in the range from 1.2 to 3.6 μg ml−1 at 465 nm and 485 nm for cyclophosphamide and ifosphamide, respectively, using 380 nm as an excitation wavelength for both drugs with accuracies of 100.4 ± 1.83% and 99.5 ± 1.64%, respectively. This condensation reaction, however, does not differentiate between the two isomeric drugs. Another new method is, therefore, suggested. It depends on the fact that the secondary amino group of ifosphamide is hindered electronically and sterically, while the nucleophilic amino group of cyclophosphamide reacts with the nitrosyl group of sodium nitroprusside molecule, giving a bluish green colour measurable at 708 nm. This colour is used for the determination of cyclophospahmide in the presence of its isomer ifophamide in the range from 0.2 to 1 mg ml−1 with repeatability of 99.2 ± 1.49%. The suggested procedures are applied successfully to pharmaceutical formulations of cyclophosphamide and ifosphamide, and their validities are assessed by applying the standard addition technique. The results obtained by applying the proposed procedures are statistically analyzed and compared with those obtained by adopting the United States Pharmacopoeial method.

Spectrofluorimetric Method for the Determination of Cyclophosphamide and Ifosphamide

Abstract Cyclophosphamide and ifosphamide are determined by fluorescence reaction based on oxidation of cyclophosphamide or ifosphamide with sodium peroxide to give the perphosphorus compound. The perphosphous compound is allowed to oxidise indole to the highly fluorescent indoxyl, equivalent to cyclophosphamide or ifosphamide, which is determined spectrofluorimetry. Beer's law is obeyed within concentrations from 5×10−5 - 2.5×10−4 mg/mL The suggested procedure has an accuracy of 99.9 ± 2.17 %. Application to some pharmaceutical preparations has shown no significant difference when compared with the B.P. (1988) method.

Environmental contamination and assessment of exposure to antineoplastic agents by determination of cyclophosphamide in urine of exposed pharmacy technicians: is skin absorption an important exposure route?

In the Netherlands, special guidelines and safety precautions were introduced about 10 y ago for preparation and administration of antineoplastic agents. However, little is known about the effectiveness of these measures. In this study, occupational exposure to antineoplastic agents of nine pharmacy technicians who were involved in drug preparation was investigated. Cyclophosphamide, 5-fluorouracil, and methotrexate accounted for 95% of the antineoplastic agents prepared; therefore, the presence of these compounds was monitored. During preparation, cyclophosphamide was detected in the air of the work environment (< 0.04-10.1 micrograms/m3). Contamination of and permeation through latex gloves were found for each of the three compounds. The uptake of cyclophosphamide was assessed by the determination of cyclophosphamide in urine. The drug was found in urine samples of six pharmacy technicians, including three persons who were not directly involved in the preparation of cyclophosphamide. The amounts excreted ranged from 0.2 to 19.4 micrograms/24 h. The results strongly suggest that inhalation is of minor importance for internal exposure, compared with other, presumably dermal, routes.

The determination of cyclophosphamide and its metabolites in blood plasma as stable trifluoroacetyl derivatives by electron capture chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of the antitumour drug cyclophosphamide and six stable metabolites in plasma of cancer patients, namely dechloroethyl-cyclophosphamide, 4-keto-cyclophosphamide, carboxy-phosphamide, alcophosphamide, nor-nitrogen mustard and the N-chloroethyl-1,3-oxazolidine-2-one, as methyl and/or trifluoroacetyl derivatives by single ion monitoring gas chromatography/mass spectrometry, mostly in the electron capture chemical ionization mode. The isolation of most metabolites was performed by solid-phase C-18 extraction in weakly acidic medium. The phosphoramide mustard isolated under these conditions decomposes readily to the nor-nitrogen mustard during derivatization. The original nor-nitrogen mustard and the chloroethyl-1,3-oxazolidine-2-one were isolated by liquid extraction with ethyl acetate in alkaline medium. Recoveries of 75-99% were measured using spiked blank plasma samples. Quantitation of metabolites in patient plasma samples was performed using two sets of calibration curves for the concentration ranges of 1-100 ng and 0.1-10 micrograms of metabolite per millilitre of original plasma.

Bioavailability of Cyclophosphamide in the CMF Regimen

Cyclophosphamide (CY) was administered to 22 breast cancer patients treated routinely according to the CMF regimen: 75 mg/m3/d x 14 d p.o. CY, 30 mg/m2 days 1 and 8 i.v. methotrexate (MTX) and 500 mg/m2 days 1 and 8 i.v. 5-Fluorouracil (5-FU). The sequence of drug administration was always the same: 1) CY, 2) MTX; and 3) 5-FU. Capillary gas chromatography was performed for determination of CY in blood. Bioavailability (F) could be determined in 14 patients since CY was also administered intravenously in the same dose. The data of systemic exposure of oral CY in the other 8 patients were matched to those of the first 14 in whom bioavailability could be determined. Mean F was 0.85 +/- 0.22 (85% +/- 22%); in 1 patient F was 0.43 (43%). Furthermore, 3 patients treated with only p.o. CY had low estimated F values: 0.45, 0.49 and 0.50. In comparing patient characteristics with pharmacokinetic data, it was concluded that age might have a predictive value for elimination half-life t 1/2 z of i.v. CY. The youngest patients showed shortest t1/2 z and were also amongst those with the lowest F. This indication requires an extension of the study as well as monitoring of CY metabolism as a function of age. For the premenopausal patients this might be of particular importance, since this group is known to be prone to benefit from chemotherapeutic treatment according to the CMF regimen.

Determination of cyclophosphamide in whole blood and plasma by reversed-phase high-performance liquid chromatography

A rapid, simple, and sensitive reversed-phase high-performance liquid chromatographic determination of the cytostatic drug cyclophosphamide in whole blood and plasma has been developed. The pre-chromatography isolation of the drug involves salting-out of acetonitrile with simultaneous extraction of cyclophosphamide from whole blood and plasma. A short column packed with 5-μm reversed-phase octadecylsilane (ODS) spherical particles was used with an isocratic elution of 5 m M potassium phosphate (pH 6.80) —acetonitrile (80:20, v/v). The cyclophosphamide was monitored at 190 nm and 0.40–0.002 a.u.f.s. At a flow-rate of 1.0 ml/min, the retention time of cyclophosphamide was ca. 9 min. The completion time for the assay was less than 20 min and the assay had a detection limit of 0.30 μg/ml. This method was used to determine the stability of cyclophosphamide in plasma at room temperature and at −10°C.

Polarographic and Colorimetric Methods for Determination of Cyclophosphamide

Polarographic and colorimetric methods for analysis of cyclophosphamide and its dosage forms were investigated. Both methods are based on the reaction of cyclophosphamide with nitrous acid. A single cathodic diffusion-controlled wave was used for dc polarographic determination of cyclophosphamide, with an accuracy of 99.98 +/- 1.09%. The wave was well defined and irreversible. By differential pulse polarographic analysis, as little as 10 ppm cyclophosphamide was determined; overall accuracy at 10-60 ppm was 100.16 +/- 0.99%. The linear relationship between absorbance for the lemon-yellow nitroso derivative and the concentration of cyclophosphamide was further used in colorimetric analysis; overall accuracy was 100.2 +/- 0.99%.

Isolation, identification and determination of cyclophosphamide and two of its metabolites in urine of a multiple sclerosis patient by high pressure liquid chromatography and field desorption mass spectrometry.

The off-line combination of high pressure liquid chromatography and field desorption mass spectrometry has been used for the simultaneous isolation, identification and determination of cyclophosphamide and two of its metabolites, 4-ketocyclophosphamide and carboxyphosphamide in urine from a patient suffering from multiple sclerosis. Cyclophosphamide and its metabolites were separated using reverse phase liquid chromatography. Field desorption mass spectrometry was employed for identification and quantification. The technique applied needs no derivatization for analysis. The limits of detection by field desorption mass spectrometry for 1, 2 and 3 are factor of about 4 X 10(3)-10(5) lower than those of a common variable ultraviolet detector. Quantitative determination was carried out using the method of stable isotope dilution with deuterated analogues of 1, 2 and 3. In a pilot study, the ratio of 1:2:3 was determined to 1:0.02:0.6. One ml of urine is sufficient for simultaneous analysis of the three compounds. The typical analysis time, including separation by liquid chromatography and field desorption measurement, is about 30 minutes.

Determination of cyclophosphamide in urine, serum and cerebrospinal fluid of multiple sclerosis patients by field desorption mass spectrometry

The levels of cyclophosphamide in samples of urine, serum and cerebrospinal fluid of multiple sclerosis patients, who had received the drug orally (4 × 100 mg/day) have been determined by field desorption mass spectrometry using the principle of stable isotope dilution. After thorough preparation of the samples, concentrations of cyclophosphamide of 10 to 60 μg/ml in urine and 200 to 400 ng/ml in serum and cerebrospinal fluid have been determined from 0.6–3 ml samples. The first quantitative data for cyclophosphamide in cerebrospinal fluid of multiple sclerosis patients could be established without the use of radioactive material. The relatively high level of the parent drug found in the fluid at the end of a 3-weeks treatment may shed some light on the mechanism of action of this drug in the treatment of the disease.

Quantitative GLC Determination of Cyclophosphamide and Isophosphamide in Biological Specimens

A method for measuring the antitumor agent cyclophosphamide was developed and applied to the determination of the drug in biological specimens. After extraction with ether, cyclophosphamide and the internal standard isophosphamide are converted to their trifluoroacetyl derivatives and assayed by GLC, using either an electron-capture or a flame-ionization detector. The minimum detectable amount is 25 pg/injection using the electron-capture detector. Linearity was found up to microgram amounts of substance, without any interference of endogenous substrates. Values from serum, urine, and liver in mice treated with a single dose of cyclophosphamide (85 mg/kg) are also reported.