2.1. Chemicals and reagents

Adenosine 5′-triphosphate disodium salt hydrate (99%), adenosine 5′-diphosphate sodium salt (98%), adenosine 5′-monophosphate sodium salt (99%), perchloric acid (99%), and tetrabutylammonium hydrogensulfate (97%) (TBAS) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Acetonitrile (Cryochrom, Russia), potassium dihydrogen phosphate anhydrous (HiMedia Laboratories Pvt. Limited, India), and potassium carbonate (Applichem, Germany) were of analytical grade.

2.2. Instrumentation

Chromatographic analyses were performed with an LC-20A system (Shimadzu, Japan) equipped with SPD-M20A UV/VIS photodiode array detector, CTO-20AC column oven, DGU-20A3 degasser, and CBM-20A communications bus module. Analytes were separated on a Luna C18 column (5 μm, 250 mm × 4.6 mm i.d.) (Phenomenex, CA, USA) provided with C18 Security Guard cartridge (5 μm, 4 mm × 3 mm i.d.) (Phenomenex, CA, USA). pH measurements were carried out with HI 2210 pH meter (Hanna Instruments, RI, USA). Shaking water bath GFL1092 (GFL, Germany), 5415R centrifuge (Eppendorf, Germany), UV-1650PC spectrophotometer (Shimadzu, Japan), microscope (LOMO, Russia), and CPX 130 ultrasonic processor (Cole-Parmer, IL, USA) were used for the bacterial cultivation and biological sample preparation.

2.3. Chromatographic conditions

The mobile phase consisted of (A) acetonitrile and (B) 50 mM monobasic potassium phosphate (pH 4.6) with 25 mM TBAS in a ratio of 0.5 (A):99.5 (B). The chromatographic separation was carried out at 25 °C and flow rate of 1 mL/min. The injection volume was 20 μL. The identification of peaks was based on the comparison of retention times and diode-array spectra of biological samples and chemical standards. ATP, ADP, and AMP were detected at 254 nm.

2.4. Standard solution preparation

Stock solutions of 10 mM ATP, ADP, and AMP were prepared by dissolving the appropriate amounts of these compounds in 10 mL of deionized water. Aliquots of the standard solutions were kept at −20 °C. The working solutions were prepared daily by appropriate dilutions with deionized water.

2.5. Bacterial strains and growth conditions

Experiments were performed with M. smegmatis strain mc²155 (ATCC Number: 700084).

A loop of M. smegmatis culture maintained on Middlebrook 7H10 agar (Difco, France) plate was inoculated into 5 mL Middlebrook 7H9 broth (Difco, France) supplemented with Tween 80 (0.05%) and ampicillin (50 μg/mL) and cultivated at 37 °C and 200 rpm for about 36 h. The resulting culture of approximate concentration of 10⁸ CFU/mL was diluted (1:200) in 50 mL of fresh Middlebrook 7H9 broth supplemented with Tween 80 (0.05%) and cultivated at 37 °C and 200 rpm. Aliquots of the culture for the analysis were sampled at time specified in the text below.

The culture growth was monitored by the absorbance at 600 nm.

Cell viability was tested by cell plating on Luria-Bertani agar (Sigma, USA) and counting of colony forming units (CFU) after 3 days of incubation at 37 °C.

Cell biomass was estimated by optical density (at 600 nm), preliminary calibrated in values of absolutely dry weight. The calibration curve was plotted by measuring the mass of equal volumes of cell suspensions of different optical densities deposited on filters and heated for 18 h at 105 °C.

2.6. Sample preparation

Cells were placed into 550 μL of 0.4 N HClO₄ and sonicated for 30 s (230 V, 20 kHz, amplitude 50%). The resulting suspension was centrifuged (1 min, 16,000 g, 0 °C), and the supernatant was neutralized with 2 M K₂CO₃ (pH 7.0). All manipulations were performed on ice. Neutralized samples were stored at −20 °C and then thawed and centrifuged (1 min, 16,000 g, 25 °C) before HPLC analysis.

2.7. Microscopy and smear preparation

Bacterial smears stained with gentian violet (15 min) or carbolic fuchsin (Ziehl-Neelsen method) were viewed using oil immersion microscopy (1000× multiplication; not less than 20 fields were examined).

3.1. Extraction of nucleotides

A thick cell wall of M. smegmatis makes difficulties for the intracellular component extraction. Treatment of such kind of bacteria with acid is less effective as compared to gram-negative bacteria, and the boiling extraction procedure usually takes more than 5 min [5, 23]. Here, we proposed a rapid method of extraction based on sonication of cells in perchloric acid. The completeness of cell destruction was estimated microscopically and by the viability test. The treatment of bacterial cells during 30 s resulted in a complete destruction of cell walls (Figure 1) and allowed an entire release of cell components into media. In agreement with, no colony forming units were detected in the cell suspension after sonication. The similar results were obtained after sonication of 24 hour old culture (5 × 10⁶–10⁷ CFU/mL) (depicted in Figure 1), 48 hour old culture (5 × 10⁸–10⁹ CFU/mL), and 72 hour old culture (5 × 10⁸–10⁹ CFU/mL) (data not shown). Less time of sonication in perchloric acid or an application of the influencing factors separately did not lead to the desired effect. It should be noted that the usage of perchloric acid was of importance since it causes inactivation and precipitation of proteins.

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Effect of sonication in 0.4 N HClO₄ on Mycobacterium smegmatis cells. A, C — control cultures without treatment; B, D — cells sonicated for 30 s. A, B — smears stained with gentian violet; C, D — smears stained with carbolic fuchsin

Citation: Acta Chromatographica Acta Chromatographica 31, 1; 10.1556/1326.2017.00344

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The influence of the extraction procedure on adenine nucleotide stability was tested in 100 μM standard solution. Variation of the nucleotide amount after the treatment was within the uncertainty of the method, specifically 98.73 ± 0.79% for ATP, 100.85 ± 0.94% for ADP, and 100.83 ± 0.79% for AMP against 100% of the untreated nucleotide solution (data presented as mean ± SD of 4 replicates).

3.2. Chromatographic separation

The proposed method allowed the separation of ATP, ADP, and AMP simultaneously within 30 min (Figure 2). The adequate selectivity of adenine nucleotides was achieved by using of tetrabutylammonium hydrogensulfate as ion-pair reagent and 0.5% acetonitrile. The application of the elution solvent containing more than 0.5% acetonitrile reduced the analysis time but impaired the selectivity in early of the analysis making the determination of AMP in biological samples impossible. An increase in acetonitrile concentration up to 1–5% of the elution solvent from 10 min after the start time resulted in a decrease of the ATP retention time and an improving of its resolution. However, such a modification of the chromatographic program required additional time for equilibration of the column cancelling the time saving and reduced the selectivity of AMP and ADP in the next run.

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Separation of AMP (1), ADP (2), and ATP (3) in standard solution (A) and Mycobacterium smegmatis cell extract (B)

Citation: Acta Chromatographica Acta Chromatographica 31, 1; 10.1556/1326.2017.00344

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An increase in retention time of the target peaks, especially AMP, occurred in biological samples as compared to standard solution. This shift was observed in all extracts and seemed to be due to the presence of other compounds. The correctness of peak identification in biological samples was confirmed by the standard addition method and ultraviolet (UV) spectrum data.

3.3. Linearity and limits of quantification of the method

The linearity of the method was assayed using nine-point calibration curves representing the relation between the peak area values and concentration of the compounds. Calibration data are shown in Table 1. The limit of detection was defined as the amount of a substance that produced a detector signal that could be clearly distinguished from the baseline (6 fold higher than the baseline noise). Equations for calculating the amount of nucleotides in injection volume are presented in Table 1.

3.4. Determination of adenine nucleotide intracellular concentration in M. smegmatis

Adenine nucleotide pools were analyzed in M. smegmatis cells of different growth phases. Extraction and HPLC analysis were performed as described in Experimental section. External standard method and, in some cases, standard addition method were used for the quantification of adenine nucleotide concentration in biological samples. The amount of nucleotides in injection volume of mycobacterial extracts ranged, at an average, from 18 to 200 pmol for ATP, 10–90 pmol for ADP, and 20–100 pmol for AMP depending on the culture density (number of cells) and physiological state.

To calculate the intracellular nucleotide concentration, the obtained values of nucleotide concentration in extracts (nmol/L) were divided by the values of dry weight of the biomass which nucleotides were extracted from (mg/L). The data presented in Table 2 demonstrate an applicability of the proposed method for mycobacterium cultures of different optical density and physiological states.

Energy charge (EC) which is considered as a measure of energy stored in the adenine nucleotides and, in turn, as a parameter of the metabolic potential of cells can be calculated according to the formula: EC = ATP + 0.5ADP/(AMP + ADP + ATP) [1]. The data in Table 2 indicate that energy charge of actively growing mycobacteria was 0.7–0.8 and declined when cells enter stationary phase. The energy charge values of M. smegmatis we obtained were in agreement with those reported by Lee and Colston, who used the boiling in buffer for the extraction of nucleotides and the fluorescence method for their analysis [5].

3.5. Precision and accuracy

Precision is expressed in terms of relative standard deviation (RSD) calculated based on the values of at least 30 replications within 3 months (Table 3). Coefficient of variation for retention time involves both values in standard solutions and biological samples and demonstrates a sufficient repeatability of the method. The variation of the peak area values in standard nucleotide solution was no higher than 1.8%, but the intracellular concentration determination was less precise (4–7.5% RSD). This discrepancy could be due to the additional random errors originated from sample collection and processing.