GC-FID determination of nucleobases guanine, adenine, cytosine, and thymine from DNA by precolumn derivatization with isobutyl chloroformate
© Brohi et al. 2016
Received: 4 February 2016
Accepted: 28 March 2016
Published: 7 April 2016
Adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) are building blocks and major constituents of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The metabolic pathway of DNA and RNA is strongly related to A, G, C, T, and U.
Gas chromatography (GC) has been examined for the ease of separation of the nucleobases guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U) after precolumn derivatization with isobutyl chloroformate. GC was carried out from the column HP-5 (30 m × 0.32 mm id) with layer thickness 0.25 μm. The linear calibration curves were obtained with 0.5–50 μmol L−1 with limits of detection (LOD) 0.08–0.10 μmol L−1.
The isolated DNA after acid hydrolysis from human blood and plants was analyzed, and amounts of T, A, C, and G were found in human blood within the ranges 0.521–2.625, 0.559–2.655, 0.490–2.915, and 0.450–2.739 μmol L−1, respectively, with relative standard deviation (RSD) 0.77–5.66 %. The ratios between T:A and G:C were 0.94–1.05 and 0.94–1.06, respectively. The values of (T + A)/(G + C) were within 0.90–1.07. Similarly from plant, T, A, C, and G were found within 0.48–2.915 nmolg−1 with RSD 0.88–5.00 %.
The method was applicable for the analysis of DNA nucleobases from biological samples.
Purine (guanine (G) and adenine (A)) and pyrimidine (thymine (T), cytosine (C), and uracil (U)) are the basic constituents of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and their metabolic pathway is strongly associated with the development of DNA (Xia et al. 2009). The defects in metabolism of purines and pyrimidines result in the symptoms such as mental retardation, cardiovascular diseases, renal failure, gout, and toxemia (Sheng et al. 1991). Therefore, the determination of individual concentration of nucleobases or their ratio in DNA is important in biosciences and clinical diagnosis (Wang et al. 2002; Ba et al. 2012).
A number of analytical procedures have been proposed for the determination of nucleobases including electrochemical techniques (Ba et al. 2012; Wei et al. 2011; Huang et al. 2011; Li et al. 2013; Svorc and Kalchar 2014), high-performance liquid chromatography (Gill and Indyk 2007; Yeung et al. 2008; Graven et al. 2014), capillary electrophoresis (Ye and Jiang 2002; Rodriguez-Gomez and Carabias-Martinez 2014; Haunschmidt et al. 2008), spectroscopic methods (Amri et al. 2003; Heisler et al. 2002), chemiluminescence (Liu and Xue 2006), and mass spectrometry (Huang and Chang 2007). Capillary gas chromatography (GC) has high potential to separate closely related organic compounds in a short time. It is also free from the problem of disposing off the used solvents. However, the nucleobases are not sufficiently volatile and thermally stable to elute from the GC column. Thus, derivatization is required before their determination. The derivatization reagents used include bis(trimethylsilyl) trifluoroacetamide (Kuhara et al. 2011; Laking and Gehrke 1972); pentafluorobenzoyl chloride, pentafluorophenylsulfonyl chloride, or heptafluorobutyric anhydride (Nazareth et al 1984); N,N-tert-butyl-dimethylsilyl trifluoroacetamide (Glavin et al. 2006); and N-(tert-butyl-dimethylsilyl) N-methyl trifluoroacetamide (Horst et al. 2012). The silyl reagents are effective derivatizing reagents for nucleobases but require a nonaqueous medium for their derivatization. Isobutyl chloroformate (IBCF) has been used as a derivatization reagent for GC determination of primary and secondary low-molecular-mass aliphatic amines (Lundh and Akesson 1993); dicarboxylic acids and amino acids (Sobolevsk et al. 2004); and 4-(5-)methylimidazole (Fernando and Ferreira 1997). The derivatization is carried out in an aqueous medium. The present work examines IBCF as a derivatizing reagent for the GC determination of G, A, T, C, and U from aqueous-organic solvents. The work also optimizes the derivatization and GC elution conditions for the quantitation of nucleobases from acid-hydrolyzed DNA.
Reagents and solutions
The reagent-grade compounds G, A, C, T, and U (Sigma Chemical Co. St. Louis, USA); IBCF; chloroform and acetonitrile (Fluka, Buchs, Switzerland); methanol (Rdh Chemical Co. Spring Valley, CA); and pyridine (E-Merck, Darmstadt, Germany) were used.
Guaranteed reagent-grade hydrochloric acid (37 %), sodium bicarbonate, acetic acid, potassium chloride, sodium acetate, boric acid, sodium tetraborate, ammonium chloride, sodium carbonate, and ammonia solution (E-Merck, Darmstadt, Germany) were used.
Buffer solutions (0.2 M) between pH 1 and 12 at unit interval were prepared from the following: potassium chloride adjusted pH with hydrochloric acid (pH 1–2), acetic acid-sodium acetate (pH 3–6), ammonium acetate (pH 7), boric acid-sodium tetraborate (pH 7.5–8.5), sodium bicarbonate-sodium carbonate (pH 9), ammonia solution-ammonium chloride (pH 10), and potassium hydroxide-potassium chloride (pH 11–12).
The stock solutions (1 mmol L−1) of G, A, C, T, and U were prepared in water-methanol (1:1 V/V). Further solutions were prepared by suitable dilution.
GC studies were carried out on Agilent model 6890 network GC system, connected with flame ionization detection (FID) and split injector (Agilent Technologies, Santa Clara, CA, USA), hydrogen generator (Parker Balston, Analytical Gas Systems, H2-90, Parker Hannifin, Haverhill, USA), and pure nitrogen (British Oxygen Company (BOC), Karachi, Pakistan); computer with ChemStation software controlled the gas chromatograph.
Capillary column HP-5 (30 m × 0.32 mm id) with film thickness 0.25 μm (J&W Scientific GC column, Wilmington, NC, USA) was used throughout the study.
GC-FID analysis and sample preparation
An aliquot of solution 0.5–1.0 mL containing 0.5–50 μmol L−1 A, T, C, U, and G was added to 0.5 mL of solvent containing acetonitrile-water-pyridine-methanol (40:40:10:10 V/V/V/V), 0.5 mL carbonate buffer (pH 9), and 0.25 mL of IBCF (10 % in methanol). The mixture was mixed well and placed for 15 min in ultrasonic water bath at room temperature (30 °C). Chloroform (0.5 mL) was then added and the contents were shacked well. The two layers were then allowed to separate, and an aliquot of organic layer was pipetted out in a screw-capped sample vial. The 1-μL solution was then injected on the column HP-5 at initial column temperature 100 °C for 1 min, followed by heating rate of 30 °C min−1 up to 280 °C and hold time at maximum temperature 2 min. The temperatures for injection and detection systems were 290 °C. The split ratio was 10:1, and carrier gas (nitrogen) flow rate was 1.5 mL min−1. The FID flow rate for nitrogen as make-up gas was fixed at 45 mL min−1; air and hydrogen flow rates were 450 and 40 mL min−1, respectively.
Sample collection and sample preparation
Five blood samples were collected by vein puncture in tubes of EDTA by verbal consent at Molecular Biology Laboratory, Liaquat University of Medical and Health Sciences (LUMHS), Jamshoro. The isolation of the DNA from blood was carried out at LUMHS by the reported procedure by Ali et al. (2013), following inorganic method (Grimberg et al. 1989). An aliquot of isolated DNA was obtained and was used in the next step as received.
A total of three samples of plant leaves, one from each (cotton, rice, and jasmine), were collected, and isolation of DNA was carried out at the laboratories of Nuclear Institute of Agriculture (NIA), Tando Jam. The isolation of DNA was carried out by a slightly modified procedure reported by Choudhary et al. (Choudhary et al. 2003). Leaves (5 g) were cleaned, washed, and ground with 5 mL of cetyl trimethyl ammonium bromide (C.TAB) buffer. After grinding, the mixture was kept in an ice machine for 20–30 min and was then allowed to achieve room temperature. Five milliliters of isoamyl alcohol-chloroform mixture (1:24) was added, and contents were shacked well. The mixture was centrifuged for 30 min at 4000g. The upper layer was collected, and 5 mL of isoamyl alcohol-chloroform (1:24) was added again into it, and the process was repeated. Then, 2-propanol (5 mL) was added to the aqueous layer, and off-white threads were collected at the bottom of the tube and were centrifuged for 5 min at 1000g. The residue was dissolved in 1 mL of tris buffer.
Acid hydrolysis of extracted DNA
Perchloric acid (1 mL) was added to a 100-μL isolated DNA sample in a screw-capped tube and was heated at water bath for 1 h at 95–100 °C. The pH of the solution was adjusted to 7 with 2 M sodium hydroxide and was filtered. The solution 0.2–0.5 mL was treated as a GC-FID analytical procedure. The quantitation of the nucleobases G, C, A, T, and U was carried out from a freshly prepared external calibration curve using linear regression equation y = ax + b.
G, A, C, and T contents from acid-hydrolyzed spiked DNA samples using linear calibration
The acid-hydrolyzed DNA (0.5 mL) was taken in duplicate. A part was processed as discussed in the GC-FID procedure, and the other was added to 0.5 mL of the solution containing G, C, A, and T (1 μmol L−1) each, and again, the procedure of GC-FID was repeated. The quantitation was carried out from the external calibration curve and from the increase in response with added standard.
Results and discussion
Optimization of derivatization
Analytical method validation
Results of quantitation of purine and pyrimidine bases using isobutyl chloroformate derivatizing reagent
Purine and pyrimidine bases
Limits of detection (LOD) (μmol L−1)
Limits of quantitation (LOQ) (μmol L−1)
Calibration range (μmol L−1)
Coefficient of determination (r 2)
The presence of ascorbic acid, glucose, methylparaben, gum acacia, Na, K, Ca, Mg, Cl, CO3, SO4, and NO3 did not affect the determination of the nucleobases, when present at least twice the concentration of nucleobases.
Analysis of acid-hydrolyzed DNA of human blood and cotton, rice, and jasmine leaves for T, C, A, and G concentration
Purine and pyrimidine bases
Blood sample 1 (μmol L−1)
Blood sample 2 (μmol L−1)
Blood sample 3 (μmol L−1)
Blood sample 4 (μmol L−1)
Blood sample 5(μmol L−1)
Cotton (nmol g−1)
Rice (nmol g−1)
Jasmine (nmol g−1)
Comparison of the present study with the reported GC methods used for determination of purines and pyrimidines
Compounds determined (sample)
Analysis time (min)
Pentafluorobenzoyl chloride, pentafluorophenylsulfonyl chloride and heptafluorobutyric anhydride
C, U, and T (standards)
Fused silica capillary column, (15 m × 0.25 mm id)
10 nmol–1 μmol mL−1
Nazareth et al. 1984
T, U, C, A, and G (yeast and tobacco mosaic virus RNA, salmon sperm, and calf thymus DNA)
10 % (weight)
SE-30 on 100/120 mesh “Supelcoport” packed in a 1.0 m × 4.0 mm id glass column
2–50 μmol mL−1
Laking and Gehrke 1972
U, T, A, C, and G (calf thymus DNA)
12-m SE-30 fused silica column
0.2–20 μmol mL−1
Gelijkens et al. 1981
Uracil herbicide residues (bromacil, lenacil, terbacil) (Echinacea angustifolia Moench)
Fused silica capillary column
(15 m × 0.32 mm id, 0.25-mm film thickness)
0.1–100 mmol L−1
Tekel et al. 1998
Isobutyl chloroformate (IBCF)
T, U, A, C, and G (human blood, rice, cotton, and jasmine DNA)
Column HP-5 (30 m × 0.32 mm id), 0.25-μm layer thickness
0.5–50 μmol mL−1
A GC procedure has been developed for a complete separation and determination of nucleobases after precolumn derivatization with IBCF. The derivatization is carried out from an aqueous-organic phase. The method is sufficiently sensitive to be used for the analysis of DNA after acid hydrolysis. The method is reproducible with RSD within 6 %.
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