Detection of homogentisic acid by electrospray ionization mass spectrometry

Abstract Objective Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI‐MS). Methods We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI‐MS. Results The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. Conclusion We identified the molecular ion of HGA in all sample solutions by ESI‐MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI‐MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.

strong oxidant. 11In addition, we observed that alkaptonuric urine or HGA solution became dark brown and showed characteristic absorbance peaks following the addition of NaOH and NaOCl•5H 2 O. 11 We have also reported that the oxidation of HGA and brown color change that occur after the addition of NaOH and NaOCl•5H 2 O were inhibited by ascorbic acid (AA), which is an antioxidant. 11,12 the present study, we observed the color changes of HGA solutions under different pH conditions and examined the influences of adding alkaline solution and AA on the oxidation of HGA.Furthermore, we analyzed the HGA solutions by electrospray ionization mass spectrometry (ESI-MS).We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA.Thus, this method enables the reliable detection of HGA in urine samples obtained from patients with alkaptonuria.

| Sample preparation and observation of the color change
A solution of 10.0 g/L HGA was prepared from high-purity analytical reagents and pure water.Then, a total of 800 μL of each 1.0 g/L HGA solution (pH 6.0, 7.0, and 8.0) was prepared by mixing 80 μL of 10.0 g/L HGA and 720 μL of pH 6.0, pH 7.0, or pH 8.0 phosphate buffer solution, respectively.Next, these samples were incubated at room temperature for 24 h.For the addition of the alkaline solution, 30 μL of 0.5 M KOH was added to 800 μL of 1.0 g/L HGA (pH 6.0, 7.0, and 8.0), and these samples were incubated at room temperature for 1 h.To examine the effects of an antioxidant, 40 μL of 10.0 g/L AA was added to the mixture of 80 μL of 10.0 g/L HGA and 680 μL of pH 6.0, pH 7.0, or pH 8.0 phosphate buffer solution, followed by the addition of 30 μL of 0.5 M KOH, and these samples were also incubated at room temperature for 1 h.

| Electrospray ionization mass spectrometry (ESI-MS)
The HGA solutions were measured by Fourier transform ion cyclotron resonance mass spectrometry using SolariX 9.4 T (Bruker AA was added to the HGA solutions at pH 6.0 (I), pH 7.0 (II), and pH 8.0 (III), and then KOH was added to each sample, followed by incubation of the samples at room temperature for 1 h.
Daltonics, Bremen, Germany).Mass spectra were calibrated using external calibration with a tuning-mix (Agilent, Santa Clara, CA, USA).The following instrument parameters were used: the sample flow rate was 2 μL/min, the desolvation plate temperature was 150°C, the rate of N 2 drying gas was 2.5 L/min, the rate of N 2 nebulizing gas was 1.5 L/min, and the capillary voltage was 4.5 kV for the negative ion detection mode.

| Color changes of HGA solutions under different pH conditions
First, we observed the color changes of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0).The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h (Figure 2A).In contrast, the HGA solutions at pH 6.0 and 7.0 were transparent and color changes were not recognized before or after incubation at room temperature for 24 h (Figure 2A).Next, we added the KOH, which is a strong alkali, to the HGA solutions (pH 6.0, 7.0, and 8.0) and observed the color changes.The HGA solution at pH 8.0 became dark brown after the addition of KOH (Figure 2B).However, the HGA solutions at pH 6.0 and 7.0 incubated with KOH did not show color changes (Figure 2B).

| Effect of ascorbic acid
We then investigated whether AA, an antioxidant, affected the color changes of HGA solutions (pH 6.0, 7.0, and 8.0).The HGA solution at pH 8.0 treated with AA turned a light brown color after the addition of KOH (Figure 3).Conversely, the HGA solutions at pH 6.0 and 7.0 treated with AA did not show color changes (Figure 3).

| ESI-MS analysis
We analyzed the chemical reaction of HGA (C 8 H 8 O 4 M.W. 168.15) solutions by ESI-MS (Figure 4).The HGA solution at pH 6.0 showed a deprotonated molecular ion peak [M-H] − at m/z 167.035 in the negative ion mode (Figure 4A), and after incubation at room temperature for 24 h, it still showed an ion peak at m/z 167.035 [M-H] − (Figure 4B).Furthermore, the HGA solution at pH 6.0 showed the same ion peak at m/z 167.035 [M-H] − after the addition of KOH (Figure 4C).Similar deprotonated molecular ion peaks were obtained for the HGA solutions at pH 7.0 and 8.0 In addition, we analyzed the reaction of HGA solutions treated with AA (C 6 H 8 O 6 , M.W. 176.12) by ESI-MS (Figure 5).

| DISCUSS ION
In the present study, we observed the changes in the color and mass Previous studies reported that HGA consumed more oxygen under alkaline conditions. 8,9Therefore, we examined the color change of the HGA solution at pH 6.0, 7.0, and 8.0.Although the HGA solutions under acidic and neutral conditions (pH 6.0 and 7.0) showed no color change, the HGA solution under the alkaline condition (pH 8.0) turned dark brown after incubation at room temperature for 24 h.This color change is likely caused by the oxidation of HGA to benzoquinone acetic acid (BQA). 7,13We have also reported that the HGA solution and alkaptonuric urine became dark brown after the addition of an alkali, owing to the oxidation of HGA to BQA. 11 Herein, we observed the color changes of HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) after the addition of KOH, a strong alkali.The HGA solution at pH 8.0 became darker brown following the addition of KOH.In contrast, the HGA solutions at pH 6.0 and 7.0 did not show color changes.Notably, the pH of urine is influenced by daily diet. 14,15sed on these results, when the urinary pH of patients with alkaptonuria is less than 7 (from neutral to acidic condition), it may be difficult to observe the brown color change, even if an alkaline solution is

F I G U R E 1 F I G U R E 3
Structures of homogentisic acid (HGA) and benzoquinone acetic acid (BQA).F I G U R E 2 Color change of HGA solutions.(A) HGA solutions at pH 6.0 (I), pH 7.0 (II), and pH 8.0 (III) after incubation at room temperature for 24 h.(B) HGA solutions at pH 6.0 (I), pH 7.0 (II), and pH 8.0 (III) after incubation with KOH at room temperature for 1 h.Color change of HGA solutions treated with AA.
The HGA solution at pH 6.0 treated with AA exhibited the deprotonated F I G U R E 4 ESI-MS spectra of HGA solutions.(A) HGA solution at pH 6.0.(B) HGA solution at pH 6.0 after incubation at room temperature for 24 h.(C) HGA solution at pH 6.0 after incubation with KOH at room temperature for 1 h.(D) HGA solution at pH 7.0.(E) HGA solution at pH 7.0 after incubation at room temperature for 24 h.(F) HGA solution at pH 7.0 after incubation with KOH at room temperature for 1 h.(G) HGA solution at pH 8.0.(H) HGA solution at pH 8.0 after incubation at room temperature for 24 h.(I) HGA solution at pH 8.0 after incubation with KOH at room temperature for 1 h.molecular peak [M-H] − of HGA at m/z 167.035 and that of AA at m/z 175.025 (Figure 5A).The HGA solution at pH 6.0 treated with AA after the addition of KOH also exhibited the deprotonated molecular ion peak [M-H] − of HGA at m/z 167.035 and that of AA at m/z 175.025 (Figure 5B).Similar deprotonated molecular ions were obtained for the HGA solutions at pH 7.0 and 8.0 treated with AA (Figure 5C-F).
spectra of HGA solutions.The deprotonated molecular ion peaks of HGA were detected in all sample solutions by ESI-MS, regardless of different pH conditions (pH 6.0, 7.0, and 8.0), color changes, or the presence of AA.These results may contribute to the development of HGA detection methods in urine samples obtained from patients with alkaptonuria.

F I G U R E 5
ESI-MS spectra of HGA solutions treated with AA. (A) HGA solution at pH 6.0 was treated with AA. (B) HGA solution at pH 6.0 was treated with AA after incubation with KOH at room temperature for 1 h.(C) HGA solution at pH 7.0 was treated with AA. (D) HGA solution at pH 7.0 was treated with AA after incubation with KOH at room temperature for 1 h.(E) HGA solution at pH 8.0 was treated with AA. (F) HGA solution at pH 8.0 was treated with AA after incubation with KOH at room temperature for 1 h.