HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Abstract

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

Keywords:

• bleomycin fractions
• hydrophilic interaction chromatography
• pharmaceuticals
• plasma
• quadrupole-time of flight mass spectrometry
• quality drug control

Notes

^a M—molecular weight of bleomycin A2 Cu complex (C₅₅H₈₃N₁₇O₂₁S₃Cu).

^b M—molecular weight of bleomycin B2 Cu complex (C₅₅H₈₃N₂₀O₂₁S₂Cu).

^a Concentrations of bleomycin A2 and B2 fractions in standard substance of bleomycin are calculated from their area ratio on the assumption that their detection responses per 1 mg/mL are equal (see Section “QTOF-MS Detection” and Table 1) and the sum of the A2 and B2 peak areas is considered as the maximum (i.e., 100%) content of bleomycin fractions A2 and B2 in the sample (with no impurities). The relative content of bleomycin in the sample, however, can be further influenced by unidentified impurities and/or other active as well as nonactive (minor) forms of bleomycin^{[ 3 ]} in the sample (unidentified peaks in TIC profiles). The saline solution spiked with the bleomycin standard was used to obtain the performance parameters data.

^a Concentrations of bleomycin A2 and B2 fractions in (i) Bleomedac (c = 200 ng/mL) and (ii) plasma spiked with standard bleomycin (c = 1000 ng/mL) are calculated from their area ratio on the assumption that their detection responses per 1 mg/mL are equal (see Section “QTOF-MS Detection” and Table 1).

^b The saline spiked with the standard bleomycin (Table 2) served as the reference (i.e., 100% bleomycin content). The reference peak area was compared with the tested (Bleomedac) one and the resulting value (i.e., determined bleomycin content) is stated here.

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