Development of a high performance liquid chromatographic (HPLC) method for the detection of bleomycin A2 and B2 in human plasma

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CHAPTER 3 A rapid High-Performance Liquid Chromatographic (HPLC) method for the detection of bleomycin A2 and B2 in human plasma


Bleomycin is a well-known chemotherapeutic drug used in the treatment of squamous cell carcinomas, testicular cancer and malignant lymphomas. 1 It is devoid of myelotoxicity and cardiotoxicity, and does not cause diarrhea, vomiting or nausea. 2 Its anti-neoplastic effect is via oxidant damage to DNA. 3
The major side effect of bleomycin following intravenous administration is the development of pulmonary fibrosis. 4,5 Alveolar capillary membrane damage due to free radical formation has been cited as one of the factors resulting in the development of pulmonary fibrosis.6 It is for this very reason that bleomycin is widely used to induce lung injury in various animal models with resultant oxidant-induced inflammation and fibrotic lesions in the lung interstitium.3 Although bleomycin-induced pulmonary toxicity is generally considered to be a dose related side-effect, it has been documented that pulmonary fibrosis can occur with any bleomycin dosage. 6
Bleomycin has more recently, been employed to treat haemangiomas of infancy, with very good results. 1,7,8 However, concerns about the use of chemotherapy to treat benign tumours and the possible development of bleomycin induced pulmonary fibrosis in such patients remain. Indeed, the plasma concentration of bleomycin following intralesional injection into vascular lesions is unknown. The monitoring of bleomycin levels in body fluids following intralesional therapy is imperative for the establishment of safety parameters for its use.
Various analytical methods have been developed to assay bleomycin in biological fluids. 2-6,13 Broughton and Strong in 1976 used a radioimmunoassay method to assay this compound in phosphate buffered saline (PBS) and in serum. 5 However, bleomycins are a mixture of active fractions (A1-A6; B1-B5), and the pharmacology of the different composite fractions are clinically important.
Furthermore, clinically administered bleomycin (bleomycin sulphate USP, or Blenoxane™ in the case of this study) consists of, by weight, 55-70% bleomycin A2, 25-32% bleomycin B2, and the remaining percentage divided among the other sub-fractions.
This radioimmunoassay method was inadequate as it did not distinguish between the various components of the bleomycin mixture. 13 Back in 1979, Shiu et al. motivated for the development of an assay method for all the major components of bleomycin. 2
In 1980 Shiu and Goehl published a high performance liquid chromatography (HPLC) method for the specific determination of one of the major component of the bleomycin mixture, namely bleomycin A2, in plasma. 6
Ten years later another group developed a more sensitive HPLC method using a fluorescence detector in a linear gradient, ion-paired reversed phase procedure to assay bleomycin A2 in human plasma and rat hepatocytes. 14 These HPLC methods for the determination of bleomycin in plasma were validated for the A2 fraction only. Furthermore, these long assay methods would not be optimally applicable for monitoring a large number of patients’ plasma samples.
In the present study, a rapid high performance liquid chromatographic method for the separation and quantitation of both major fractions, bleomycin A2 and B2, in human plasma was developed. This method was then employed to determine levels of bleomycin in patients treated with intralesional bleomycin.

Materials and Method


Bleomycin A2 (BWS-18) was donated by the National Institute of Health, Japan, and Bleomycin B2 (BMT 049 B2), was donated by Nippon Kayaku, Co, Ltd. (Japan). Methanol and acetonitrile were of HPLC grade; acetic acid was of reagent grade (Radchem, Johannesburg, SA). Sodium heptanesulfonate was purchased from African Biotech Consultants (Johannesburg, SA). Water was purified by a MilliQ water purification system.


A Waters™ LC Module1High Performance Chromatograph (HPLC) was used. The HPLC system and conditions are summarized in Table 3.1.

Preparation of mobile phase

The mobile phase consisted of water-0.0085 M sodium heptanesulfonate: acetonitrile: acetic acid (70:25:5). MilliQ purified water used to prepare the mobile phase was 18 mega ohm quality. The final pH of the mobile phase solution was 4.7. The solution was filtered through a 0.45 nylon membrane to remove contaminants. The solution was then degassed prior to transfer to an HPLC solvent bottle. Throughout the analysis, the mobile phase was sparged with helium at a rate of 500 ml/min to prevent the formation of air-bubbles, which can cause a considerable drop in backpressure.
For the assay of bleomycin fractions, a mobile phase flow rate of 1 ml/min was established. The resulting backpressure was approximately 2000 psi. The temperature of the column was maintained at 40oC during separation.

Bleomycin Stock

Bleomycin stock solutions were prepared at 0.8 mg/ml bleomycin A2 and 1 mg/ml bleomycin B2 in 0.1 M sodium-phosphate buffer, pH 6.8. The stock solutions were kept at -20OC until use. Standard solutions of bleomycin fractions were prepared from stock solutions to calibrate the system prior to the assay of samples.

Preparation of standards

Frozen stock solutions were thawed and diluted with milli-Q water to obtain a range of concentrations (1, 2, 4, 6, 8, 200, 250, 300 µg/ml bleomycin A2; 2, 4, 6, 8, 10, 300, 350, 400 µg/ml bleomycin B2) required for the preparation of plasma standards. Standards were prepared by adding 10 µl of bleomycin A2 or B2 to 100 µl of drug free plasma. Hence, the corresponding plasma standards were 0.1, 0.2, 0.4, 0.6, 0.8, 20, 25, 30 µg/ml bleomycin A2; 0.2, 0.4, 0.6, 0.8, 1, 30, 35, 40 µg/ml bleomycin B2. Levels above 160 µg/ml bleomycin were measured in the patient samples. Therefore another standard curve was created at concentrations of 100, 200, 400, 600, 800 µg/ml A2; 100, 200, 400, 600, 800, 1000 µg/ml B2. The system was calibrated daily with the standards prior to analysis to account for interday variations in experimental conditions.

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Assay procedure

The samples were vortexed for 30 seconds and then centrifuged for 10 minutes at 3000 rpm. The supernatant was filtered twice through a 0.2 µm cellulose acetate filter. For each dilution, 20 µl of the filtered plasma sample was injected on to the column.

Validation of the assay method

Validation is the presentation of documented evidence that all causes for variation have been accounted for, and that any variation present will not be excessive of expected variation or standard curve variation. 14 The main analytical variables for the validation of a HPLC method are accuracy, linearity, specificity, precision and sensitivity.15


The accuracy of an analytical procedure expresses the closeness of agreement between a value which is accepted either as a conventional true value or an accepted true value and the measured value. 14 In this study, the accuracy of the method was determined by comparing the peak heights resulting from spiked plasma standards, and the peak heights obtained from direct injection of the same amount of drug in aqueous solution.


The linearity of an analytical procedure is the ability (within a given range) to obtain test results that are directly proportional to the concentration of analyte in the sample. 14 Linearity was established for 5 concentrations across the range of the analytical procedure. The linearity of this assay was assessed by comparison of calibration curves from analyses of spiked samples of bleomycin A2 at 0.1 to 0.8 µg/ml and of bleomycin B2 at 0.2 to 1.0 µg/ml on three different days.


Specificity is the ability to unequivocally assess the analyte in the presence of components that are expected to be present. Identification tests were performed by injecting each entity separately into the HPLC.


The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. 14
Aliquots of plasma were spiked with known amounts of the bleomycin stock solution to give a range of concentrations (table 3.3). The plasma samples were assayed in triplicate. The mean, standard deviation, the coefficient of variation (CV) and relative error for the assay were calculated.

Limit of detection (LOD)

The Detection limit is the lowest amount of analyte in a sample which can be detected, but not necessarily quantitated as an exact value. 14 The LOD was based on the standard deviation of the response curve and the slope, and was expressed as follows: LOD = 3. 3σ where, σ = the standard deviation of the response S = the slope of the calibration curve

Limit of quantitation (LOQ)

The quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. 14 The LOQ was based on the standard deviation of the response curve and the slope, and was calculated as follows:
LOQ = 10σ  where, σ = the standard deviation of the response
S = the slope of the calibration curve

Patient s

In this study which was approved by the University of Pretoria ethics committee (IPA19-96/2000), blood samples were obtained from ten patients with vascular anomalies treated with bleomycin. Informed consent was obtained from all patients or guardians (in the case of children younger than 18 years). Intralesional bleomycin was used for the treatment of four patients with haemangiomas at dosages of 0.2-0.94 mg/kg/therapy. Patient information is tabulated in table 3.4.

List of figures
List of tables 
CHAPTER 1 General Introduction
1.1. Motivation for the study
1. 2. Purpose of investigation
1.3. Objectives
CHAPTER 2 Literature Review
I. Bleomycin
2.1. Chemistry of bleomycin
2.2. Chemical structure
2.3. Metal ion coordination
2.4. Mechanism of biological activity
2.4.1. Effect of bleomycin on DNA
2.4.1. Effect of bleomycin on RNA
2.4.1. Effect of bleomycin on proteins
2.5. Metabolism of bleomycin
2.6. Side Effects.
2.7. Clinical use
II. Haemangiomas
2.8. Introduction
2.9. Nomenclature
2.10. Natural History
2.11. Complications
2.12. Pathophysiology
2.13. Treatment
III. Angiogenesis concept
2.14. Introduction
2.15. Growth factors in angiogenesis
2.15.1. Vascular endothelial growth factor
2.15.2. Basic fibroblast growth factor
2.16. Apoptosis
2.17. Mitomycin C
2.18. The Cell Cytoskeleton
CHAPTER 3 Development of a high performance liquid chromatographic (HPLC) method for the detection of bleomycin A2 and B2 in human plasma
3.1. Introduction
3.2. Materials and Methods
3.3. Results.
3.4. Discussion
CHAPTER 4 The effects of bleomycin, mitomycin C, and multiple cytoskeletal-disrupting agents on endothelial cell migration and growth 
4.1. Introduction.
4.2. Materials and Methods
4.3. Statistical Analysis
4.4. Results
4.5. Discussion
CHAPTER 5 Bleomycin, mitomycin C, and cytoskeletal-disrupting agents induce endothelial cell apoptosis 
5.1. Introduction
5.2. Materials and Methods
5.3. Results
5.4. Discussion
CHAPTER 6 The effects of bleomycin on in vitro angiogenesis
6.1. Introduction
6.2. Materials and Methods
6.3. Statistical Analysis
6.4. Results
6.5. Discussion
CHAPTER 7 Effects of angiogenesis inhibitors on vascular tumour growth in an animal haemangioma model
7. 1. Introduction
7. 2. Materials and Methods
7. 3. Data Analysis
7.4. Results
7.5. Discussion
CHAPTER 8 Conclusion.

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