Chemiluminescence of LucigeninâAllantoin and Its Application for the

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Chemiluminescence of lucigenin-allantoin and its application for the detection of allantoin Muhammad Saqib, Baohua Lou, Mohamed Ibrahim Halawa, Shimeles Addisu Kitte, Zhongyuan Liu, and Guobao Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04271 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Analytical Chemistry

Chemiluminescence of lucigenin-allantoin and its application for the detection of allantoin Muhammad Saqib,†,‡ Baohua Lou,† Mohamed Ibrahim Halawa,†,‡ Shimeles Addisu Kitte,†,‡ Zhongyuan Liu,† Guobao Xu*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, PR China ‡ University of Chinese Academy of Sciences, No. 19A Yuquanlu, Beijing 100049, PR China ABSTRACT: Allantoin has been reported as a promising biomarker for monitoring of oxidative stress in humans and widely utilized in a variety of topical pharmaceuticals and cosmetics. Currently, the detection of allantoin is achieved by using chromatographic coupled techniques, which needs sample pre-extraction, derivatization, complex matrixes, and expensive instrumentation. Herein we report both the intense chemiluminescence of allantoin with lucigenin and the chemiluminescent detection of allantoin for the first time. The lucigenin-allantoin system demonstrated chemiluminescence emission intensity 17 times higher than that of the classic lucigenin-hydrogen peroxide system. Based on this fascinating phenomenon, a novel chemiluminescence method has been developed for the sensitive and selective allantoin determination with the combination of flow injection analysis. This method shows linear calibration curve in the range of 0.1–3000 µM with a detection limit (3σ/s) of 0.03 µM. Moreover, it was successfully utilized for the determination of allantoin in human eye drops and urine real samples after simple dilution with water. It shows excellent recoveries in the range of 94.0–101.7 % and each measurement takes very short time. This method exhibits potential advantages in the form of simplicity, rapidity, sensitivity, selectivity, and low cost. Allantoin could be an effective candidate for constructing new chemiluminescence systems, and may provide broad range of sensing applications.

INTRODUCTION Chemiluminescence (CL) is among the most sensitive analytical tools to achieve higher signal to noise ratio in several biological and chemical applications.1-3 CL assays are extensively studied due to their potential advantages such as simple, rapid, wide linear range, and because they do not require a light source, which results in a simple instrumentation and low or zero background signal.4 In 1935, Gleu and Petsch reported the CL reaction of lucigenin (N,N-dimethyl-9,9'-biacridinium dinitrate) with hydrogen peroxide (H2O2) in the presence of some metal cations for the first time.5 Since then lucigenin is a most popular luminophore.6 During last decades, several researchers have discussed the mechanism of the lucigenin CL reaction in detail. Lucigenin can react with oxidizing agents (e.g. H2O2) to generate CL signals under alkaline conditions.7 The CL of lucigenin has gained tremendous interest due to its applications in trace metal analysis, biological and enzyme assays, and in the analysis of organic reducing agents. However, the application of classical lucigenin-H2O2 CL system in sensing protocols is typically limited due to poor specificity.8 So it is highly desired to develop new lucigenin CL systems with higher selectivity to overcome the limitations and widen its applications in bioassays. Reactive oxygen species (ROS) can cause indiscriminant oxidative damage to biological molecules such as proteins, lipids, and DNA due to imbalance between their generation and elimination processes.9 The in vivo measurement of ROS is still a challenge, because these ROS are short lived and unstable

species so it is difficult to detect them directly in humans.10 The most convenient alternative strategy is the measurement of relevant biomarkers generated by the reaction between ROS and biological molecules. Considerable efforts have been made to develop bioassays for the identification and quantification for biomarkers of oxidative stress.11 Allantoin (5-ureidohydantoin), is the major product resulted from the oxidative reactions of ROS10,12 and found stable in urine.13 Notably, allantoin displays the level of ROS itself without relying on the scavenger uric acid.14 Allantoin concentration in urine is variable at least twofold with regard to the oxidative status in healthy individuals.15 Therefore, allantoin can be used as a promising biomarker for examining the oxidative stress in human. Several reports have revealed the higher level of allantoin in disorders related to oxidative stress such as inflammatory and autoimmune conditions,16 diabetes,14,15 pulmonary,17 renal,14 cardiovascular,18 and Wilson's disease.19 Furthermore, allantoin levels in urine, plasma and muscles were found useful to measure the oxidative status related to exercise.20 The level of allantoin is reported as more sensitive biomarker of oxidative stress than cysteine and glutathione concentrations.21 Allantoin also plays crucial roles in the metabolism, assimilation, storage, and transport of nitrogen in plants.22,23 A number of studies have supported the phenomenon that allantoin acts as a vitamin and in vivo antioxidant.24 Also, allantoin is the main ingredient in a wide number of medicines and cosmetics, particularly in skin care products.

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The U.S. Food and Drug Administration (FDA) have registered allantoin as an effective and safe ingredient for pharmaceutical and skin care products in the range of 0.1% to 2.0%.25,26 Therefore, it is extensively used in dermatologic and pharmaceutical products for the treatment of ulcers, burns, psoriasis, slow-healing wounds, and dry skin. It is used in variety of cosmetics including skin creams, powders, lip-care products, hair care products, suntan and sunburn lotions, mouthwashes and diaper rash ointments. Therefore, it is also highly desirable to determine the allantoin concentration in a variety of products from pharmaceutical and cosmetic industries for quality control assurance. In the past, allantoin measurement has been achieved by a colorimetric method by using Rimin-Schryver reaction,27 which was further improved with the induction of highperformance liquid chromatography (HPLC).12 The levels of urinary and serum allantoin have also been quantified with the help of Gas chromatography–mass spectrometry (GS/MS) methods.28,29 Several other analytical methods has also been documented including normal phase chromatography,30 HPLC-UV,26 capillary electrophoresis (CE),31 hydrophilic interaction liquid chromatography (HILIC),32 liquid chromatography-tandem mass spectrometry (LC-MS/MS techniques),33,34 and ultra performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS).35 However, these methods have shown variable results due to several limitations.32 The limitations in the measurements methods are still a barrier for the accomplishment of allantoin as a biomarker in oxidative stress studies. Most of the reported methods generally involved solid phase extraction procedures, derivatization steps, usage of an internal standard (isotopic), and LC or GC separation tools.36 These laborious sample procedures results in time consuming, expensive analysis, and also leads to overestimation of allantoin levels due to urate or oxidation in the sample.36 Moreover, spectrophotometric detection approaches in LC or GC protocols for allantoin have been limited by lack of specificity and sensitivity due to effects of complex matrix.34 Therefore, it would be a timely progress to develop an alternative simple, rapid, sensitive and specific method that can allow allantoin determination, both in pharmaceutical and clinical samples, and can also be used in the quality control assurance. In this study we reported, for the first time, allantoin as a stable and effective coreactant for lucigenin CL. It can efficiently react with lucigenin to generate strong CL emissions without adding additional catalysts. The lucigenin-allantoin CL system was then applied for the sensitive detection of allantoin and lucigenin; particularly it displayed good selectivity for allantoin among several potential interfering species. To the best of our knowledge, it is the first CL method reported for the allantoin detection so far. The advantages of this CL method as compared with the previous methods are better sensitivity, higher stability, reduced amount of analyte, and significantly reduced analysis time and cost associated with expensive instrumentation and techniques. EXPERIMENTAL SECTION Chemicals and reagents. Allantoin, creatinine, cysteine, glutathione, arginine, uric acid, glucose, alanine, histidine, tryptophan, methionine, glycine and tyrosine were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Lucigenin was purchased from TCI. Hydrogen peroxide was purchased from Beijing Chemical Reagent Company. Lucigenin

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stock solution (1 mM) was prepared by dissolving 0.0510 g lucigenin in 100 mL water. All the chemicals were analyticalreagent grade and were used without further purification. Double distilled water was used during all the experiments. Apparatus. The CL signals were measured by utilizing a flow injection analysis (FIA) based CL system. It consists of an IFIS-C mode intelligent flow injection sampler (ReMax Inc., Xi'an, China), a BPCL ultra-weak luminescence analyzer (Institute of Biophysics, Chinese Academic of Sciences), and a home-made flow cell. The home-made flow cell was kept in a light-tight (dark black) box of the luminescent analyzer to measure the CL signals. The loop injector was equipped with an injection loop of 50 µL to inject the target analytes.

Scheme 1. Schematic description of lucigenin-allantoin CL detection system based on the FIA. A, B represents the flow channels; C represents the IFIS-C mode intelligent flow injection sampler; D represents the loop injector; E represents the CL detector; and F represents the waste cup. Procedure of lucigenin detection. Scheme 1 shows the schematic description of this new FIA system for the detection of lucigenin. Briefly, 10 mM allantoin in 1.5 M NaOH and water were respectively pumped into the flow cell through channels A and B at a flow rate of 1.6 mL/min. Different concentrations of lucigenin prepared in water were injected through the loop injector. Procedure of allantoin detection. Scheme 1 shows the schematic description of the FIA system for the detection of allantoin. Briefly, 1.5 M NaOH and water solutions were respectively pumped into the flow cell through channels A and B at a flow rate of 0.95 mL/min. Different concentrations of allantoin were mixed with 0.1 mM lucigenin in water first, and then mixtures were injected through the loop injector. Stock solution of allantoin 10 mM was prepared by dissolving 0.0158 g solid allantoin into 10 ml double distilled water. Allantoin standard solutions of concentrations ranging from 0.1-3000 µM were prepared by diluting the stock solution with double distilled water after that the standard solutions were analyzed as described above. Procedure of sample preparation. In human eye drops samples, allantoin concentration was labeled as 0.1%. The human urine samples were collected from three healthy individuals. The eye drop and human urine samples were diluted with double distilled water to avoid the other interfering species and to bring the allantoin concentrations in the linear calibration range. The percentage recoveries of present method were calculated by simple dilution of eye drops with double distilled water and by spiking the urine real samples with different concentrations of allantoin, respectively. Each measurement was carried out in triplicate.


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RESULTS AND DISCUSSION CL of lucigenin-allantoin system. Allantoin was found to be able to efficiently react with lucigenin to generate intense CL signals, as illustrated in Figure 1. The CL intensity of the lucigenin and lucigenin-H2O2 systems was also measured, respectively (Figure 1). As shown in CL intensity-time curves relationships, lucigenin-allantoin system displayed a remarkable ~17 time increase over classic lucigenin- H2O2 system. It indicates that allantoin is a potential coreactant for CL studies. As demonstrated in Figure 2, a chemiluminescent spectrum was recorded by utilizing different wavelength filters in the range of 400-640 nm. The maximum CL emission was observed at about 490 nm (Figure 2) that is in agreement with the typical lucigenin spectrum. The proposed reaction mechanism is shown in Scheme 2. It has been reported that allantoin can decompose and can generate oxidant species in alkaline medium (Eq. (1) in Scheme 2).37,38 The cyano radical generated from the decomposition of allantoin in alkaline solutions may react with lucigenin to excite and produce the primary emitter, singlet N-methylacridone, and subsequently resulted in strong CL (Eqs. (2 & 3) in Scheme 2).39,40

Figure 1. Chemiluminescencent kinetic profiles of lucigenin (red line), lucigenin–H2O2 (blue line) and lucigenin–allantoin systems (green line). The inset focuses on enlarged CL intensity–time curve for the lucigenin (red line), and lucigenin–H2O2 (blue line). Comparison was performed in 1.5M NaOH, in the presence of 0.1mM lucigenin, 2 mM H2O2, and 2 mM allantoin. Photomultiplier tube (PMT) voltage: 800 V.

Scheme 2. Possible mechanism of lucigenin-allantoin CL detection system.

Figure 2. CL emission intensity was plotted as a function of wavelengths from 400 to 640nm. Spectrum was recorded in 1.5M NaOH, in the presence of 0.1mM lucigenin and 2 mM allantoin. PMT voltage: 900 V.

Figure 3. Chemiluminescencent kinetic profiles in the presence (blue line) and absence (red line) of oxygen. The measurement was performed in 1.5M NaOH, in the presence of 0.1 mM lucigenin and 2 mM allantoin. PMT voltage: 600 V. Effect of oxygen. It has been reported that the oxidation of lucigenin by dissolved oxygen can generate CL signals in alkaline medium. Thus, the measurements were performed in the absence and presence of oxygen to record its effect on CL intensities of the lucigenin-allantoin system. Before the reaction, N2 gas was bubbled through the lucigenin and allantoin solutions for 30 min to remove the dissolved oxygen. As shown in Figure 3, CL intensity decreased negligibly after the removal of dissolved oxygen from the reactant solutions. It reveals that dissolved oxygen is not responsible for the CL emission of the lucigenin-allantoin CL system. Unlike previous lucigenin CL systems, the proposed system showed negligible interference from the dissolved oxygen. It is particularly advantageous in CL detection to avoid the interference from oxygen. Effect of NaOH on CL. By understanding the basic mechanism of lucigenin oxidation, highly alkaline conditions are preferable for effective CL emission. It is well documented that the concentration of NaOH has significant effect on the-



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Table 1. Comparison of different methods used for the detection of allantoin. Analytical method Linear range RP- LC-UV method Enzyme cycling method HPLC–UV/Vis method GC-MS method HPLC–UV method HPLC‐APCI‐MS method HPLC- UV/Vis method IP-HPLC–UV method HILIC-UV method HILIC-MS/MS method CE–UV method UVDS method CE-UV method Lucigenin-allantoin CL method

0.69–128.9 µM 0–70 µM 0.25–50.0 µM 5.0–50 µM 2.0–8.0 µM, 10.0–50.0 µM 116.92–3792 µM 0.5−50.0 µM 2345.7−9408.2 µM 37.92–88.48 µM 1.58−118.5 µM 0.5–150 µM 316–1896 µM 6.32−1264 µM 0.1−3000 µM

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LOD

Ref.

0.06 µM ------0.5 µM 0.111 µM 1.0 µM 10.68 µM 0.15 µM 0.58 µM 4.42 µM 0.47 µM 0.3 µM ------3.16 µM 0.03 µM

41 30 14 29 42 43 44 45 46 47 31 48 49

Present work

RP= Reverse Phase, LC= Liquid Chromatography, UV= Ultra Violet, Vis= Visible, HPLC= High Performance Liquid Chromatography, GC= Gas Chromatography, MS= Mass Spectrometry, APCI= Atmospheric Pressure Chemical Ionization, IP= Ion Pair, HILIC= Hydrophilic Interaction Liquid Chromatography, CE= Capillary Electrophoresis, UVDS= Ultraviolet Derivative Spectrophotometry, CL= Chemiluminescence.

- CL intensities of lucigenin. The solubility of allantoin is also increased in the presence of NaOH. Previous studies have also stated that NaOH could be a suitable basic medium to decompose allantoin into detectable species.36 Moreover, lucigeninallantoin CL intensity increases rapidly in the presence of NaOH. Therefore, we tested CL intensities of lucigeninallantoin system in different concentrations of NaOH solutions. Figure 4 shows that the CL intensity increases rapidly in the NaOH concentration range of 0.01 to 1.5 M, and level off above this range. From the above results, it can be proposed that the increase in CL intensity with increasing NaOH concentration up to 1.5 M is mainly due to the fast production of oxidant radical species (cyano radicals) from allantoin and lucigenin deprotonation process. Therefore, 1.5 M NaOH is selected for the further experiments due to highest CL intensity and faster reaction speed. Detection of lucigenin. The proposed CL system was then applied for the sensitive detection of lucigenin. As demonstrated in Figure 5, the CL profiles have good linear relationship over lucigenin concentrations from 0.01-100 µM. Linear equation is obtained as logarithm (log) I = 2.34 + 0.79 log c with a correlation coefficient “r” of 0.994 (c refers to the concentrations of lucigenin in µM). With the help of this novel method, the limit of detection (LOD) is achieved as low as 7 nM for lucigenin detection according to 3σ/s method (where σ is the standard deviation of the blank (n=9), and s is the slope of the corresponding calibration curve). In comparison with the previous reports, our method emerges as simple and more sensitive method of lucigenin detection.50 The relative standard deviation (RSD) is 2.61% for nine consecutive measurements (n=9) at lucigenin concentration of 0.1 µM, which indicates the good reproducibility of proposed method. Detection of allantoin. This new CL system was also employed for the highly sensitive detection of allantoin. Figure 6 displays the CL response of the lucigenin-allantoin system as a function of different concentrations of allantoin. It is evident that the CL intensities increased with the increase of the concentration of allantoin. The log of CL enhancement response was linear with the log of concentrations of allantoin in the range of 0.1 to 3000 µM. The linear equation is expressed as log I = -0.24 + 0.42 log c (r = 0.994).

Figure 4. (A) CL emission–time curves in different concentrations of NaOH from 0.01 to 2 M and (B) CL intensity was plotted as a function of the concentration of NaOH. The measurements were performed in the presence of 0.2 mM lucigenin and 2 mM allantoin. PMT voltage: 800 V.


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Figure 5. (A) CL emission–time curves in different concentrations of lucigenin from 0.01 to 100 µM and (B) Linear relationship between log of CL intensity and log of concentration of lucigenin. The detection was performed in 1.5M NaOH, in the presence of 10 mM allantoin. PMT voltage: 1000 V. LOD (3σ/s) is calculated to be 0.03µM for allantoin. A survey of the literature shows that this value (LOD) is better than the reported values for allantoin.25,32 By comparison, it is simple, rapid, sensitive and has wide linear range than most of the previously reported methods for the detection of allantoin (Table 1). Moreover, it is the first CL method for the detection of allantoin. This CL method achieves reproducible results with a RSD of 3.45% for nine successive detections at concentration of 3 µM (Figure 7). Selectivity of this method for allantoin. The selectivity of the proposed method for the detection of allantoin was evaluated by measuring the relative CL response toward some common interfering biological compounds and metal ions. Figure 8A shows the relative CL response of 0.1 mM of creatinine, cysteine, glutathione, arginine, uric acid, glucose, alanine, histidine, tryptophan, methionine, glycine and tyrosine on the detection of allantoin. The concentrations of all biological compounds used for interference studies are higher than that in normal medicinal/real samples. In eye drops samples, some metal ions such as ZnSO4 are also present.

Figure 6. (A) CL emission–time curves in different concentrations of allantoin from 0.1 to 3000 µM and (B) the log of CL enhancement efficiency (I-I0)/I0was plotted as a function of log of concentrations of allantoin. I0 and I represent the CL intensity in the absence and presence of allantoin, respectively. The detection was performed in 1.5M NaOH, in the presence of 0.1 mM lucigenin. PMT voltage: 1000 V.

Figure 7. CL emission-time curves at the allantoin concentration of 3 µM. The measurement was performed in 1.5 M NaOH, in the presence of 0.1 mM lucigenin. PMT voltage: 1000 V.



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fold to avoid the interference from potential interferants. The allantoin concentrations in urine samples were calculated as 2.39 ± 0.87 µM by standard addition method, which are comparable to literature values for allantoin in human healthy controls.32 To calculate the recoveries, urine samples were then spiked with 1, 5, and 10 µM known concentrations of allantoin, respectively. As shown in Table 2, the proposed method shows excellent recoveries in the range of 94.01– 101.7% and each measurement takes very short time for completion. The obtained results shows that this method is suitable for the real sample applications. Table 2. Analytical results for the determination of allantoin in eye drops samples. Samples

Eye drops

Urine

a

Figure 8. Selectivity of the proposed method in the presence of (A) biological compounds and (B) common metal ions. The concentrations of the biological compounds and metal ion are 0.1mM and 10µM, respectively. The measurements were performed in 1.5M NaOH, in the presence of 0.1 mM lucigenin and 0.1 mM allantoin. PMT voltage: 1000 V. Therefore, the interference effect of typical metal ions including Na+, Zn2+, Fe2+, Pb2+, Ni2+, Cu2+, Mn2+, Fe3+, Cd2+, Cr3+, and Co2+ on this new CL method was also investigated. Figure 8B demonstrates that, the relative CL increase in CL intensities is negligible with the addition of a number of metal ions at higher concentrations (10 µM). The above results reveal that these substances have no significant effects on allantoin determination, indicating that this method has good selectivity among several potential interferents. Determination of allantoin in practical samples. To check the practical applicability of present method, the determinations of allantoin in human eye drop and human urine samples have been carried out. Eye drops samples were purchased from the local pharmacy and drug store in Changchun, Jilin province, China. To evaluate the recoveries, eye drops samples were simply diluted with double distilled water to bring the labelled concentrations up to 1, 5, and 10 µM for allantoin, respectively. The human urine samples were collected from three healthy individuals and were diluted 10-

Concentrations of allantoin Amount Amount Amount claimeda added foundb (µM) (µM) (µM) 1.00 --0.94

Recovery (%)

RSD (n=3;%)

94.01

1.91

5.00

---

4.83

96.60

2.75

10.0

---

9.78

97.81

3.40

---

0.00

2.39

---

2.90

---

1.00

3.34

98.52

2.65

---

5.00

7.40

100.1

3.01

---

10.0

12.6

101.7

3.50

The concentrations labeled on the eye drops samples. b The average of three replicate determinations.

CONCLUSIONS We have reported, for the first time, the reaction of allantoin with lucigenin to generate intense CL without adding any other luminescent materials or catalysts. This fascinating CL phenomenon in combination with FIA provides a facile method for sensitive and selective allantoin determination with good linearity and reproducibility. Also, it has been successfully applied for the determination of allantoin in the human eye drop and urine real samples without derivatization and separation technique. The proposed CL method is simple, cheap, and fast and has high sensitivity, good selectivity, and acceptable reproducibility. Since allantoin is an important biomarker for oxidative stress and with broad applications in medicinal and cosmetics industries, our method may find wide applications in clinical analysis, quality assurance and other chemical and biological sensing.

AUTHOR INFORMATION Corresponding Authors * Tel: +86-431-85262747. Fax: +86-431-85262747. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This project was kindly supported by the National Key Research and Development Program of China (No. 2016YFA0201300), the National Natural Science Foundation of China (Nos. 21475123 and 21505128), the Chinese Academy of Sciences (CAS)–the


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Academy of Sciences for the Developing World (TWAS) President’s Fellowship Programme (2013-053).

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