Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA

Abstract

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10(9) nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

A rapid, sensitive and solvent‐less method for determination of malonaldehyde in meat by stir bar sorptive extraction coupled thermal desorption and gas chromatography/mass spectrometry with in situ derivatization

“…Several more specific measures have been used, including high-performance liquid chromatography ultraviolet (HPLC-UV)/fluorescence, [7] gas chromatography (GC)/MS, [8] and LC/MS methods, [9] with different derivatization under milder conditions, particularly with reactive hydrazine compounds, such asmethylhydrazine, 2, 4-dinitrophenylhydrazine in urine, [10] 2,4,6-trichlorophenylhydrazine in plasma, [11] phenylhydrazine in plasma, [12] and pentafluorophenylhydrazine (PFPH) in urine and cell culture. [13,14] The main advantage of these methods is the milder derivatization conditions, followed by more specific analysis. However, most of the methods need to concentrate and extract the derivatives of MDA from complex matrices.…”

“…Therefore, the derivatization conditions are critical for determination of MDA levels in complex food matrices and in biological samples. Several more specific measures have been used, including high‐performance liquid chromatography ultraviolet (HPLC‐UV)/fluorescence, gas chromatography (GC)/MS, and LC/MS methods, with different derivatization under milder conditions, particularly with reactive hydrazine compounds, such asmethylhydrazine, 2, 4‐dinitrophenylhydrazine in urine, 2,4,6‐trichlorophenylhydrazine in plasma, phenylhydrazine in plasma, and pentafluorophenylhydrazine (PFPH) in urine and cell culture . The main advantage of these methods is the milder derivatization conditions, followed by more specific analysis.…”

Plant Uptake and Metabolism of Nitrofuran Antibiotics in Spring Onion Grown in Nitrofuran-Contaminated Soil

“…Isotope-labeled compound 4 ([ 13 C, 15 N 2 ]-4) was synthesized by reacting [ 13 C, 15 N 2 ]-4′ with 5-nitro-2-furaldehyde, as described previously. 25 Using a similar strategy, the 2-nitrobenzaldehyde derivative of [ 13 C, 15 N 2 ]-4′ was also synthesized by reacting [ 13 C, 15 N 2 ]-4′ with 2-nitrobenzaldehyde.…”

“…High-performance liquid chromatography (HPLC)-grade acetonitrile, ethyl acetate, hexane, and methanol were obtained from Tedia (Fairfield, OH). Isotope-labeled compound 4 ([ 13 C, 15 N 2 ]- 4 ) was synthesized by reacting [ 13 C, 15 N 2 ]- 4′ with 5-nitro-2-furaldehyde, as described previously . Using a similar strategy, the 2-nitrobenzaldehyde derivative of [ 13 C, 15 N 2 ]- 4′ was also synthesized by reacting [ 13 C, 15 N 2 ]- 4′ with 2-nitrobenzaldehyde.…”

A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

“…In previous ARP-based fluorescence and colorimetric assays, the detection limit was estimated to be approximately 1-2 AP sites per 10 4 nucleotides [16,21,22], which was not independently verified by LC-MS. Because 70 ng DNA sample was used in the assay, the detection limit translates to about 15 fmol AP sites [16]. The sensitivity of our ECL sensor is significantly higher than the fluorescence and colorimetric assays, and is also slightly higher than the value of LC-MS/MS method (6.5 fmol) [55].…”