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Colorimetric Microtiter Plate Assay of Polycationic Aminoglycoside Antibiotics in Culture Broth Using Amaranth.

We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR3-). Under acidic conditions, at which NEO exists in fully protonated form (NEOH6+), the AR3- anion associates with the NEOH6+ cation to form a precipitate, NEOH(AR)2. The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR3- anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.


Introduction
Aminoglycosides and aminocyclitols comprise a well-known and important class of clinically valuable antibiotics that interact with rRNA and inhibit protein biosynthesis. Streptomycin was reported as the first major aminoglycoside in 1944, and thereafter, novel aminoglycosides continued to be reported into the 1990s. 1 In the 21st century, however, the discovery rate of novel compounds using traditional screening techniques has been extremely low. Previous methods for the determination of aminoglycosides have included spectroscopy, 2-4 chromatography, 5,6 antibiotic activity assay, and immunoassay, 7,8 but new approaches suitable for high-throughput screening will be needed to detect additional aminoglycosides.
In previous papers, we have presented a colorimetric method for the determination of the natural polyamine ε-poly-L-lysine based on precipitation of the analyte in polycationic form with colored anions, such as 12-molybdosilicate 9,10 and dipicrylamine. 11 This method was applied successfully to screening not only for the synthetic enzyme for ε-poly-L-lysine but also for streptothricin, 12,13 which is a polycationic antibiotic possessing several amino groups. Neomycin (NEO) has six amino groups (Fig. 1a), and exists as a hexavalent cationic species (NEOH 6+ ) by protonation under certain acidic conditions. Therefore, we studied the precipitation reaction of aminoglycosides with various colored anions, and found that a reddish anionic dye, the amaranth (AR 3-; Fig. 1b) anion, forms analytical reagent for the colorimetric determination of a specific aminoglycoside.
However, it was found that the precipitation reaction was considerably affected by electrolyte anions. Therefore, a separation pretreatment is needed for the colorimetric determination of NEO in culture broth. In a previous paper, we presented a method for the separation of polyamines from culture broth using the tetraphenylborate (TPB -) anion. 14 This method was based on precipitating the polyelectrolyte salts of the analyte in polycationic form with the TPBanion, washing the precipitate with organic solvent to purify the polyelectrolytes, and re-precipitating the hydrochloride salts. In this study, the method was optimized for the separation of NEO from culture broth. The introduction of the pretreatment made it possible to detect NEO as well as an intermediate in the NEO-producing culture broth.
The production of NEO by Streptomyces fradiae NBRC12773 was performed using a slight modification of the previously described method. 15 S. fradiae NBRC12773 was cultivated in culture medium containing 2% (w/w) starch, 0.5% glucose, 0.5% yeast extract, 0.3% peptone, 0.3% meat extract, 0.02% KH2PO4, 0.06% MgSO4·7H2O, and 0.1% CaCO3 for 5 days at 28 C with rotary shaking. The culture broth was centrifuged, and proteins and non-polar compounds were removed from the resulting supernatant using chloroform extraction. The resulting sample was then subjected to further experiments.

Measurements
The absorption spectra were recorded by a JASCO V-630 spectrophotometer with a pass length of 1 cm. In the microplate assay, the absorbance was recorded with a Bio-Rad iMark microplate reader.
The NEO-producing culture broths and the pretreated samples were analyzed by HPLC/ESI-MS (Esquire 4000; Bruker) using a reverse-phase column (Sunniest RP-AQUA, 3 μm, 100 × 2.1 mm; ChromaNik Technologies) at 30 C at a flow rate of 0.3 mL min -1 with an initial gradient of 5% acetonitrile in water to 15% acetonitrile over 5 min, which was then ramped to 50% acetonitrile over 10 min. Both the acetonitrile and water contained 0.05% n-heptafluorobutyric acid and 0.05% formic acid.

Microtiter plate assay
The microtiter plate assay of NEO was performed as follows. 1. A 50-μL aliquot of the test solution was transferred into the well. 2. The test solution was mixed with 10 μL of the 1 mol/L MES and 0.5 mol/L NaOH buffer (pH 6.2) and 50 μL of the 0.3 mol/L NaTPB aqueous solution; the mixture was centrifuged, and the supernatant was removed to obtain a mixed precipitate containing an NEOH n+ -TPBsalt. 3. The precipitate was mixed with 100 μL of acetonitrile (AN); the mixture was centrifuged, and the supernatant was removed to purify the NEOH n+ -TPBsalt. 4. The precipitate was mixed with 5 μL of the 5 mol/L HCl solution and 200 μL of AN; the mixture was centrifuged, and the supernatant was removed to obtain an NEOHCl6 precipitate. 5. The precipitate was mixed with 200 μL of AN; the mixture was centrifuged, and the supernatant was removed to wash the NEOHCl6 precipitate. 6. The precipitate was mixed with 100 μL of the 0.05 mol/L CH3COOH and 0.05 mol/L CH3COONa buffer (pH 4.7) and 25 μL of the 3.3 mmol/L Na3AR solution; the mixture was centrifuged, and the supernatant was removed to obtain an NEOH(AR)2 precipitate. 7. The precipitate was mixed with 100 μL of water; the mixture was centrifuged, and the supernatant was removed to wash the precipitate. 8. The precipitate was mixed with 100 μL of the 0.1 mol/L TAPS and 0.05 mol/L NaOH buffer (pH 8.5), and the absorbance at 520 nm of the resulting NEOH(AR)2 solution was measured by the microplate reader. The procedure is illustrated in Fig. 2.

Color reaction for NEO using amaranth
In this section, the standard NEO solutions with different concentrations, cNEO, of 0.15x g/L (as trisulfate salt, x = 0 -2), that is, cNEO = x mEq/L, were used as test solutions. Here, Eq/L denotes the equivalent concentration with reference to NH2-groups, that is, the charge number of the aminoglycoside in fully protonated form. Also, a 1-mL volume of test solution was examined in a centrifugation tube (15 mL) to detect the precipitate consisting of NEO by visual observation.
In the present colorimetric method, first, a 1-mL aliquot of test solution was mixed with 0.1 mL of the 0.5 mol/L CH3COOH and 0.5 mol/L CH3COONa buffer (pH 4.7), and with 0.1 mL of the 10 mEq/L (3.3 mmol/L) Na3AR aqueous solution; the mixture was centrifuged, and the supernatant was removed to obtain the precipitate. It is noted that this operation corresponds to the operation-6 in the microtiter plate assay (Fig. 2). In the case of amaranth, Eq/L denotes the equivalent concentration with reference to the charge number of the AR 3anion.
When cNEO = 0.5 mEq/L, a reddish precipitate was obtained. The pKa-value of the amino groups of NEO have been reported to be 5.7 and 7.6 -8.8. 16 Thus, at pH 4.7, NEO would exist in the fully protonated form, NEOH 6+ , according to where NEO 0 denotes the free base form of NEO. As described below, the precipitate was identified as a 1:2 NEOH 6+ -AR 3electrolyte salt, NEOH(AR)2. Therefore, the precipitation reaction can be written as Second, the precipitate was mixed with 1 mL of water; the mixture was centrifuged, and the supernatant was removed. Even after this operation, the reddish precipitate remained in the centrifugation tube, suggesting that the NEOH(AR)2 precipitate can be washed with water.
Third, the NEOH(AR)2 precipitate was mixed with 4 mL of the 0.1 mol/L TAPS and 0.05 mol/L NaOH buffer (pH 8.5). After the centrifugation, no precipitate was observed in the mixture, suggesting that the precipitate was completely dissolved in the alkaline solution. This was due to the deprotonation of some NH3 + -groups of the NEOH 6+ cation. It must be noted that, in this section, the volume of the TAPS buffer solution was four fold larger than that of the test solution, because of the high absorption coefficient of the AR 3anion.
The 0.5 mEq/L NEO standard solution gave a reddish TAPS buffer solution, the absorption spectrum of which is shown by curve (a) in Fig. 3. An absorption maximum was observed at around 520 nm, and the absorbance, A520, was determined to be A520 = 0.89 ± 0.01. The spectrum was identical to that for the 0.125 mEq/L Na3AR, 0.1 mol/L TAPS, and 0.05 mol/L NaOH solutions, indicating the presence of ~0.125(= 0.5/4) mEq/L AR 3in the TAPS buffer solution. Therefore, it can be considered that the precipitation reaction of Eq. (2) occurred quantitatively. At pH > 9, the absorption maximum shifted to a shorter wavelength and the absorbance decreased. This was attributed to the deprotonation of the phenolic hydroxyl group of amaranth. Thus, the TAPS buffer (pH 8.5) was used in this operation. In Fig. 4, the A520-value is plotted against cNEO. The A520 was proportional to cNEO at cNEO < 1 mEq/L, and remained a constant value at cNEO > 1 mEq/L, indicating again that the precipitation reaction occurred quantitatively.
The 0.5 mEq/L NEO standard solution was examined with different pHs in the first operation of the colorimetric method. In the range of pH < 6, at which NEO would exist in the form of penta-and/or hexavalent cationic species, the A520 remained at almost the same value. The A520-value decreased sharply at 6 < pH < 7.5, and became nearly equal to zero at pH > 7.5. Therefore, the AR 3anion may not precipitate the NEO species with a smaller charge number, i.e., NEOH n+ (n < 5 or 6).

Color reaction for other aminoglycoside antibiotics
The 0.5 mEq/L TOB standard solution also gave a reddish TAPS buffer solution, the absorption spectrum of which is shown by curve (a) in Fig. 5. The spectrum is identical with curve (a) in Fig. 3, indicating that the fully protonated TOB (TOBH 5+ ) was precipitated with the AR 3anion quantitatively to form TOBH(AR)5/3. The 0.5 mEq/L GEN test solution gave almost the same results as the 0.5 mEq/L NEO and TOB solutions. On the other hand, no significant precipitate was formed with the 0.5 mEq/L streptomycin, kanamycin, and amikacin (Fig. S2, Supporting Information) standard solutions. Under acidic conditions, these aminoglycoside antibiotics would  exist as tri-and tetravalent cationic species. Thus, the larger charge number may be an important factor for the precipitation of aminoglycosides with the AR 3anion.
Under the acidic condition, putrescine, spermidine, and spermine would exist as di-, tr-, and tetravalent cationic species. The 0.5 mEq/L spermidine and putrescine solutions gave no precipitate with the AR 3anion.
On the other hand, the 0.5 mEq/L spermine solution gave the reddish TAPS buffer solution, the spectrum of which is identical to that given by the 0.5 mEq/L NEO solution. The larger charge number may be an important factor also for the precipitation of the straight-chain polyamines with the AR 3anion. However, since spermine was not separated by the pretreatment with TPBanion, and would not be detected by the present microtiter plate assay. Chitosan oligosaccharides 17 and nourseothricin (a mixture of streptothricin 12,13 F, E, and D) samples would give polycationic species under the acidic condition. However, the 0.5 mEq/L chitosan oligosaccharides and nourseothricin solutions gave no precipitate with the AR 3anion.
In addition, the higher-molecular-weight polyamines, such as ε-poly-L-lysine and poly(allylamine), formed precipitates with the AR 3anion. However, the precipitate gave higher A520values than predicted, suggesting that the composition of the precipitate is complex. Cationic polymers containing quaternary ammonium groups, such as poly(diallyldimethylammonium chloride) and poly[oxyethylene(dimethylimino)propyl-(dimethylimino)ethylene chloride] 18 were precipitated with the AR 3anion, but the precipitates were not dissolved in the basic solution.

Other precipitate reagent
Evans Blue, a blue anionic dye, has a structure similar to the AR 3anion, and has four SO3groups. The 2.5 mmol/L Evans Blue solution was used instead of the 10 mEq/L Na3AR solution in the first operation of the colorimetric method. When cNEO = 0.5 mEq/L, a blue precipitate was obtained. The precipitate did not dissolve in the TAPS buffer, but dissolved in the 0.1 mol/L CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) and 0.05 mol/L NaOH buffer (pH 10.4). The absorption spectrum of the CAPS buffer solution showed an absorption maximum at around 585 nm. However, the absorbance was much smaller than that for the 0.125 mEq/L Evans Blue solution.
In the first operation of the colorimetric method, 1 mL of aqueous solution containing ~0.8 mEq/L 12-molybdosilicate 9,10 was used instead of 0.1 mL of the 10 mEq/L Na3AR solution.
When cNEO = 0.5 mEq/L, a yellow precipitate was obtained. Although the yellow precipitate was dissolved in alkaline solutions, the solutions remained colorless due to the hydrolysis of the molybdosilicate anion. Also, the NEOH 6+ cation was not precipitated by the addition of solutions containing 10 mEq/L bis(2-ethylhexyl) phosphate and sulfosuccinate anions 17 and dipicrylamine anion. 11,13 Thus, the AR 3anion can be considered to be a superior analytical reagent for NEO, TOB, and GEN, and their analogues.

Influence of electrolyte anion in the test solution
The 0.5 mEq/L NEO and cE mmol/L NH4Cl solution, where cE is the electrolyte concentration, also gave a reddish TAPS buffer solution. However, the A520-value decreased slightly with cE, as shown by plot (a) in Fig. 6. The A520-values did not change when NH4Cl was replaced by NaCl and KCl, indicating that the Clanion affects the precipitation of the NEOH 6+ cation with the AR 3anion. The NEOH 6+ cation probably associates with the hydrophilic anion to form an ion pair that is stable in aqueous solution. As shown by plot (b) in the figure, the A520value at cNEO = 0.5 mEq/L was also decreased by the addition of KH2PO4 to the test solution.
As shown by plot (c) in Fig. 6, the A520-value decreased remarkably with the addition of (NH4)2SO4. When cE > 50 mM, no precipitate was formed in the first operation, suggesting a strong interaction between NEOH6 6+ and SO4 2ions and/or the formation of a more stable ion pair. Sometimes, culture media contain high concentrations of (NH4)2SO4 as a nitrogen source. We therefore considered that a separation process was needed for use with the present colorimetric determination of the aminoglycosides in culture broth.

Separation of NEO using TPB -
In the present separation method, first, a 0.5-mL volume of test solution was mixed with 0.1 mL of the 1 mol/L MES and 0.5 mol/L NaOH buffer (pH 6.2) and with 0.5 mL of the 0.3 mol/L NaTPB solution; the mixture was centrifuged, and the supernatant was removed. This operation corresponds to the operation-2 in the microtiter plate assay (Fig. 2). In this section, we examined a model solution containing cNEO mEq/L NEO and the components in NEO-producing culture medium, 2%(w/w) starch, 0.5% glucose, 0.5% yeast extract, 0.3% peptone, 0.3% meat extract, 0.02% KH2PO4, and 0.06% MgSO4·7H2O. The 1 mEq/L NEO model solution gave a large volume of white precipitate, which was likely a mixed precipitate containing the polycationic NEO with the TPBanion. It is well known that the TPBanion forms precipitates with the K + and NH4 + ions and protonated amines. 19,20 Therefore, the precipitate would be expected to contain the salts of the monovalent cations with the TPBanion. However, these 1:1 electrolyte salts were removed in the next operation. Also, it is noted that the 1 mEq/L NEO standard solution did not yield a significant precipitate. However, the precipitation of a polycationic NEO with the TPBanion would be expected to be facilitated by the precipitation of other cationic species.
Second, the precipitate was mixed with 1 mL of AN; the mixture was centrifuged and the supernatant was removed. The amount of precipitate became much smaller, suggesting that the polycationic NEO salt with the TPBanion can be washed by this operation.
Third, the precipitate was mixed with 0.05 mL of the 5 mol/L HCl solution and with 2 mL of AN; the mixture was centrifuged and the supernatant was removed. By mixing with HCl, the TPBanion was decomposed to benzene and triphenylborane, 21 which are soluble in AN. On the other hand, NEO was precipitated as the hydrochloride salt, NEOHCl6. Finally, the precipitate was mixed with 2 mL of AN; the mixture was centrifuged, and the supernatant was removed to wash the NEOHCl6 precipitate.
The separation method was evaluated by chromatography. Figure 7A shows a chromatogram for the 1 mEq/L NEO standard solution. A well-developed elution peak was observed at around 13.3 min. The MS spectrum of the eluted substance showed a signal at m/z = 615 [(M+H) + ]. Figure 7B shows a chromatogram for the 1 mEq/L NEO model solution. Although NEO was detected from the model solution, some impurity peaks and significant background were observed in the chromatogram. From a 0.5-mL aliquot of the model solution, an NEOHCl6 precipitate was obtained, and was re-dissolved in 1 mL of water. The aqueous solution gave the chromatogram shown in Fig. 7C. The elution peak for NEO was observed, and the yield was calculated to be 89% from the peak area. Although the solution conditions in the separation process were modified, the yield was not increased. However, no remarkable impurity peak was observed.
The aqueous solution (1 mL) prepared from a 0.5 mL of the 1 mEq/L NEO model solution was assayed by the present colorimetric method. The aqueous solution gave a reddish TAPS buffer solution, the spectrum of which is shown by curve (b) in Fig. 3. The A520 was determined to be 0.78 ± 0.03, and the yield was calculated to be 88%, which is close to that obtained above. The 1 mEq/L TOB model solution also gave a white precipitate, which was assayed by the present colorimetric method. Curve (b) in Fig. 5 shows the spectrum of the resulting TAPS buffer solution. The A520-value was 0.72 ± 0.01. The 1 mEq/L GEN model solution gave A520 = 0.45 ± 0.01. The 1 mEq/L kanamycin model solution gave a white precipitate. However, as described earlier in this paper, kanamycin would not be detected by the colorimetric assay with AR 3anion. Thus, the polycationic aminoglycosides, such as NEO, TOB, and GEN, can be detected from the model solutions.

Microtiter plate assay of NEO
The microtiter plate assay was performed with the model solutions having different cNEO values and containing 2%(w/w) starch, 0.5% glucose, 0.5% yeast extract, 0.3% peptone, 0.3% meat extract, 0.02% KH2PO4, and 0.06% MgSO4·7H2O. It is noted that NEO concentrations up to 5 mEq/L are capable of being determined under the solution conditions described in Fig. 2. The resulting TAPS buffer solutions are shown in Fig. 8. The model solution of cNEO = 0 gave a colorless TAPS buffer solution, and the redness increased remarkably with cNEO. Thus, although a part of NEO would be lost during the separation process, even the 0.1 mEq/L level of NEO can be detected by visual observation. The absorbance at 520 nm for the TAPS buffer solution in the well, A′520, was determined with the plate  reader. As shown in the figure, the A′520-value increased linearly with cNEO in the range tested. The regression line is given as A′520 = (0.56 ± 0.05)(cNEO/mEq L -1 ) + (0.08 ± 0.04), with a mean square of errors 23 of 3.3 × 10 -4 . The detection limit was calculated to be 3(3.3 × 10 -4 ) 1/2 /0.56 = 0.1 mEq/L (~90 μg/mL, as trisulfate salt), indicating again that the 0.1 mEq/L or 100 μg/mL level of NEO can be determined.
NEO-producing culture broth S. fradiae was cultivated in the culture medium. As shown in Fig. 9A, the production of NEO was confirmed by the chromatogram of the culture broth. The produced amount was estimated to be ~0.85 mEq/L. The separation operation was performed using the culture broth. The resulting precipitate gave the chromatogram shown in Fig. 9B. A well-developed peak was observed earlier than that for NEO (12.8 min). The eluted substance showed a signal of m/z = 616, suggesting that the substance is an intermediate of the NEO biosynthesis, 6′′′-deamino-6′′′-hydroxyneomycin C (Fig. S3, Supporting Information). 24 This would exist as a pentavalent cationic species.
The culture broth was assayed by the present colorimetry with the separation process. Using the calibration curve of Fig. 8, the produced amount was calculated to be 1.06 ± 0.05 mEq/L, which is somewhat larger than that determined by chromatography. The positive error might be due to the existence of the intermediate. In fact, the intermediate was detected from the NEOH 6+ -AR 3precipitate. This underscores the importance of detecting not only the final products but also the intermediates in biosynthetic studies.
In conclusion, the present colorimetric method with the separation process should be useful for the biosynthetic analysis of polycationic aminoglycosides.