Simultaneous determination of pentostatin and 2‘-amino-2‘-deoxyadenosine in fermentation broth by high performance liquid chromatography-tandem mass spectrometry

Abstract

An analytical method was established for the simultaneously determination the pentostatin and 2'-amino-2'-deoxyadenosine contents in fermentation broth by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After high-speed centrifugation, aqueous solution dilution, vortex shock, and microfiltration, the fermentation broth samples were analyzed by HPLC-MS/MS. The samples were separated on a Waters Atlantis® T3 column (100 mmx2. 1 mm, 5 µm) using a gradient elution program with 10 mmol/L ammonium formate (containing 0. 1% formic acid) and methanol (containing 0.02% formic acid) as the mobile phases. Moreover, a chromatographic protection column (5 mmx2. 1 mm, 5 µm) was added to preserve the column efficiency. The flow rate, column temperature, and injection volume were set at 0. 3 mL/min, 25, and 10 µL, respectively. Qualitative and quantitative ana lyses of the target compounds were performed using an ESI+ source. MS parameters such as the collision energies and tube lens offsets of pentostatin and 2'-amino-2'-deoxyadenosine were optimized. The quantitative ion pairs of pentostatin and 2'-amino-2'-deoxyadenosine were m/z 269. 17> 153. 20 and m/z 267. 00 > 136. 10, respectively; the corresponding collision energies were 11 V and 18 V. The external standard method was used for quantitative analysis. The established method was verified rigorously in terms of the linear range, limit of detection, limit of quantification, recovery rate, and precision. Pentostatin and 2'-amino-2'-deoxyadenosine showed good linear relationships in the range of 1.0-250 µg/L. The correlation coefficients ranged from 0. 996 9 to 0. 999 6, and the relative standard deviations (RSDs) ranged from 6. 51% to 8. 35% (n = 8). This result indicated good accuracy and exactitude in the detection of the pentostatin and 2'-amino-2'-deoxyadenosine. The recoveries (n = 6) at three spiked levels (1.0, 5. 0, and 25 µg/L) were in the ranges of 97. 94%-104. 46% and 89. 96%-107. 21% for the pentostatin and 2'-amino-2'-deoxyadenosine, respectively; the corresponding RSDs were in the ranges of 3. 74%-4. 88% and 4. 81%-13. 29%. The limits of detection (LODs, S/N >3) and limits of quantification (LOQs, S/N>10) of the 2'-amino-2'-deoxyadenosine and pentostatin in the fermentation broth were 0. 003-0. 060 µg/L and 0. 010-0. 200 µg/L, respectively. The validated experimental method was used for the detection of actual samples, viz. the stored multiple pen-tostatin-producing mutagenic strains in our laboratory. The HPLC-MS/MS method for the determination of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth offered the advantages of small sampling volume, strong maneuverability, good stability, and high sensitivity. Compared with previously published methods, this systematically established and optimized method significantly reduced the detection time, and matrix effects were well suppressed. Moreover, the peak shape and stability of the target compounds were greatly improved. This method provides a methodological basis and meaningful reference for the detection of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth.