Synchronous Fluorescence Spectroscopy and Its Applications in Clinical Analysis and Food Safety Evaluation Yao-Qun Li, Xiu-Ying Li, Ali Abbas Falih Shindi, Zhe-Xiang Zou, Qian Liu, Li-Rong Lin, and Na Li
Abstract Synchronous fluorescence spectroscopy (SFS) plays an important role in the simultaneous analysis of multicomponent samples due to the remarkable advantages of spectral simplification, light scattering reduction, and selectivity improvement over conventional fluorescence spectroscopy. The selectivity and resolution are further enhanced by the combination of synchronous fluorescence approaches with other techniques, such as derivative technique, low-temperature technique, and chemometrics. In this chapter, the methodologies of various SFS approaches are compendiously introduced. Representative examples of biological, environmental, and clinical applications of these techniques are briefly summarized. An emphasis is placed on the development of rapid and simple synchronous fluorescence-based approaches for the determination of carcinogenic polycyclic aromatic hydrocarbons in foods and the differential analysis of porphyrins in human body samples.
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Introduction
Fluorescence spectrometry, possessing high sensitivity as well as its relatively low cost, is an important technique in analytical chemistry [1]. Small quantities of the analyte can be detected directly by spectrofluorometry. However, this technique has not been widely used for the simultaneous and direct determination of multicomponent samples due to the spectral overlapping problems. The resolution of the spectra of such type of mixtures becomes impossible without time-consuming separation
Y.-Q. Li (*) • X.-Y. Li • A.A.F. Shindi • Z.-X. Zou • Q. Liu • L.-R. Lin • N. Li Department of Chemistry and Key Laboratory of Analytical Sciences, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China e-mail:
[email protected] C.D. Geddes (ed.), Reviews in Fluorescence 2010, Reviews in Fluorescence, DOI 10.1007/978-1-4419-9828-6_5, © Springer Science+Business Media, LLC 2012
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Fig. 1 Different hypothetical fluorescence spectra and their corresponding states of the excitation (ex) and emission (em) monochromators; (1) fluorescence excitation spectrum; (2) fluorescence emission spectrum; and (3) synchronous fluorescence spectrum. The rectangular bar and the arrows represent the monochromator status of being fixed or scanned, respectively
process and highly expensive equipment. In order to overcome this difficulty, several techniques have been developed, such as the synchronous fluorimetry, the derivative technique, and the low-temperature technique. Recently, the combination of synchronous fluorescence spectrometry with chemometrics has been developed. Fluorescence measurements are usually carried out as follows: the excitation spectrum is obtained by scanning the excitation monochromator while setting the emission monochromator at an optimal wavelength. Similarly, an emission spectrum is generated by the scanning of the emission monochromator with the excitation monochromator set at a suitable single-excitation wavelength (Fig. 1). Synchronous fluorescence spectroscopy (SFS), first developed by Lloyd in constant-wavelength mode [2], made a milestone in fluorescence analysis. Compared to excitation and emission spectra, synchronous fluorescence spectra are obtained by scanning both the excitation and emission monochromators simultaneously rather than one of them during the measurement, which leads to some remarkable advantages, such as spectral simplification, bandwidth narrowing, reduced scattering interference, and improved resolution. SFS provides an efficient tool to increase analytical selectivity, yet maintains the fluorimetric high sensitivity. Thus, SFS has become an attractive alternative for the simultaneous determination of multiple compounds complex samples [3]. In this chapter, we provide a concise review on the different approaches of SFS and their combination with other analytical techniques. And representative examples of biological, environmental, and clinical applications of these techniques are briefly summarized. An emphasis is placed on the development of rapid and simple synchronous fluorescence-based approaches for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAHs) in foods and the differential analysis of porphyrins in human body samples.
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Synchronous Fluorescence Spectroscopic Approaches
Depending on the scanning rates of the emission and the excitation monochromators (lem and lex) and the type of the maintained difference between them, several synchronous fluorescence techniques have been developed. In conventional synchronous fluorescence spectroscopy known as constant-wavelength SFS (CWSFS), a constant wavelength difference is maintained between the excitation and emission monochromators [4]. Another variant of the synchronous fluorescence technique is constant-energy SFS (CESFS), proposed by Inman et al. [5]. In CESFS, the excitation and the emission monochromators are scanned synchronized so that a constant energy difference is maintained between two monochromators. Considerable improvements of selectivity in the analysis of PAHs mixtures were made with this technique [6]. Another approach is the fluorescence measurement carried out through the simultaneous scanning of the excitation and the emission monochromators at different rates. This approach is called variable-angle SFS (VASFS), and is known by its flexibility in addition to high selectivity [7, 8]. Besides the approaches described above, it is possible to make use of a cut in the total fluorescence spectrum. The cut can be obtained by joining points of equal intensity to produce a scan trajectory, isopotential trajectory. This spectrofluorimetric technique is known as matrix isopotential SFS (MISFS) [9]. We ever discussed the synchronous fluorescence spectrometric methodology by proposing feasible and straightforward calculations, which provide a guideline for the experimental design of synchronous fluorescence spectrometric methods in the wavelength domain [10].
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Constant-Wavelength Synchronous Fluorescence Spectroscopy
CWSFS is a traditional SFS which consists essentially of the simultaneous scanning of both the excitation and emission monochromators while maintaining a constant wavelength offset (Dl = lem − lex = constant) between two monochromators. Compared with the conventional fluorescence technique, CWSFS is capable of eliminating the Rayleigh scattering interference by scanning parallel to Rayleigh scattering ridge [11] to provide sharply peaked and narrow profiles and simplified spectrum, as shown in Fig. 2. In the application of synchronous fluorescence in a constant-wavelength mode, the Dl selection is extremely important, which always affects the profile of the spectrum, the bandwidth, and the signal intensity. The synchronous contour plot of total fluorescence data has been used to optimize the selection of Dl. The data collected by CWSFS can be represented as a line in the total fluorescence plane [12]. Owing to its remarkable advantages, CWSFS has been widely used for the determination of multicomponent mixtures [13], including PAHs, amino acids, medicines, and so on. A total of 13 PAHs in a mixture of 16 EPA PAHs in a solution of hexane
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Fig. 2 Fluorescence spectra of anthracene: (A.a) excitation spectrum, (A.b) emission spectrum, and (B) CWSF spectrum by using a Dl = 3 nm
were determined by using second-derivative constant-wavelength synchronous spectrofluorometry [14]. A simple and specific approach was established for the simultaneous determination of riboflavin and pyridoxine by CWSFS. Under the experimental conditions, two peaks appeared at 526 and 389 nm in the obtained synchronous fluorescence spectrum, corresponding to riboflavin and pyridoxine, respectively [15].
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Constant-Energy Synchronous Fluorescence Spectroscopy
Inman and Winefordner described CESFS firstly [16], by which a constant energy difference (Dn) between excitation and emission monochromators was maintained as each was scanned through the spectral region of interest in environmental samples [17, 18]. Dn was expressed as: æ 1 1 ö Dν = ç ´ 10 7. è λ ex λ em ÷ø
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Besides the advantages of CWSFS, CESFS possesses an additional advantage of the reduction of Raman scatters [3, 19]. By keeping a Dn different from Raman scattering energy DnR, Raman scattering did not appear in CESFS spectra [11]. If an appropriate Dn value is chosen during constant-energy synchronous scanning, in other words, when the energy of excitation and emission happens to match the transition between absorption and emission, a fluorophore will exhibit vibration energy transition, in which synchronous fluorescence signals reach the highest intensity with better sensitivity. As indicated in Fig. 3, by choosing different Dn, the spectral profiles are different. As for PAHs, a Dn of 1,400 cm−1 is often used in the
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CESF analysis. Take benzo(a)pyrene (BaP) as an example; the profile of BaP in dimethylsulfoxide using a Dn of 1,400 cm−1 is simpler and with higher intensity than that using a Dn of 2,370 cm−1. Considerable improvements in the analysis of polynuclear aromatic hydrocarbon mixtures have been made with this technique due to their definite vibrational energy levels [5, 20]. For the simultaneous determination of carbazole, anthracene, and perylene, CESFS has demonstrated to be a better approach than CWSFS in simplification of the scan procedures [21].
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Variable-Angle Synchronous Fluorescence Spectroscopy
The variable-angle synchronous fluorescence spectra are obtained by simultaneously scanning the monochromators (excitation and emission) at different speeds; thus, the wavelength difference between them is not constant. Compared to conventional SFS, VASFS can make full use of excitation and emission information of molecules and provides better selectivity and flexibility. The spectral resolution of this method can be further improved through the combination with derivative technique or low-temperature technique. As long as the wavelength distance between the selected VASFS path and the second-order scattering path is kept far enough, the VASF spectrum would not have second-order scattering interference. Therefore, VASFS could be used to eliminate the influence of second-order scattering in a spectrum [11]. Figure 4 shows a selection of scanning path for the application of VASFS for a hypothetical system of three components. VASFS has been widely applied in the simultaneous and rapid screening of multicomponent samples due to its better selectivity and flexibility than other approaches [8, 22, 23].
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Fig. 4 VASF scanning for a three-component hypothetical mixture of fluorophores: analyte (solid contour lines); interferents (dashed and dash-dotted contour lines). The scanning path is indicated by the straight solid line
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Matrix Isopotential Synchronous Fluorescence Spectroscopy
MISFS, first proposed by Murillo-Pulgarin [24], is a novel technique for resolving badly overlapped mixtures. The principle of MISFS is to make an isopotential trajectory by joining points of equal intensity of a compound on the fluorescence contour plot. Scanning along the isopotential trajectory gives a constant fluorescence signal of the compound. By using the derivate technique, the signal of the compound turns out to be zero. If the matrix of a sample is treated as the interfering component, derivative MISFS is capable of removing the interference from the matrix. Figure 5a shows theoretical contour map of a theoretical matrix and a theoretical analyte, and the selected isopotential trajectory. When a spectrofluorometer scans along the selected isopotential trajectory T, a total signal consisting of a signal generated by the analyte, indicated as Signal H in Fig. 5b, and a constant signal generated by the matrix, shown as Signal K in Fig. 5b, is obtained. Hence, the firstderivative fluorescence spectrum of the sample at the selected isopotential trajectory corresponds exactly to the matrix isopotential synchronous fluorescence spectrum of the analyte. The sensitivity that can be achieved in the direct determination of the analyte can be maintained by the selection of the isopotential trajectory in such a way that it passes through the emission and excitation maxima of the analyte. MISFS has been applied for the determination of the individual components in different and important matrices [25, 26]. Murillo-Pulgarin and Alanon-Molina [27] determined the individual components of the binary mixtures of salicylic and gentisic acids by MISFS.
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Combination of Synchronous Fluorescence Spectroscopy with Other Techniques Derivative Technique
Derivative technique, with the characteristics of excellent band narrowing and signal enhancement for minor spectral features, has been widely used in analytical chemistry to improve the selectivity of measurements. By applying the derivative technique, it becomes possible to convert the small shoulder exhibited by the analyte into a signal peak that can be quantified. Since SFS techniques are able to simplify the spectra, narrow the bandwidth, and provide better resolution, the combination of derivative techniques and SFS approaches provides synergistic effects on better resolution and selectivity. John and Soutar [28] first proposed the combination of the SFS and the derivative technique to get derivative SFS, which was used to develop simple, rapid, and sensitive spectrofluorimetric methods for the simultaneous analysis of mixtures with closely spaced bands [29, 30]. First-derivative CWSFS was developed for the rapid determination of carbendazim in lentinula edodes. As indicated in Fig. 6, the derivative technique provided the amplification of fluorescence signals and gave a higher resolution from background signal than direct SFS [31]. The combination of MISFS and the first-derivative technique provided good analytical results and allowed the simultaneous determination of diflunisal and salicylic acid in human serum [32]. Derivative MISFS was developed to determine the 1-hydroxypyrene in urine without preseparation [33]. We firstly combined derivative technique with CESFS and VASFS approaches to accomplish various goals. Derivative nonlinear VASFS (NLVASFS) was proposed for the simultaneous and rapid screening of PAHs in water samples. The proposed method is simple, fast, and straightforward based on a single spectrum containing all the characteristic peaks of
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Fig. 6 Constant-wavelength synchronous (dashed line) and derivative constantwavelength synchronous (solid line) fluorescence spectra of 0.2 mg/mL carbendazim at Dl = 20 nm (Reproduced from Ref. [31])
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four PAHs via a single spectral scanning of a sample [34]. Second-derivative CESFS offered a highly sensitive and selective spectrofluorimetric approach for the rapid determination of trace BaP in drinking water and in solutions leached from disposable paper cups [35].
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Low-Temperature Technique
The broadband nature of fluorescence spectra obtained at room temperature restricts the effective use in multicomponents, PAHs, for example, exhibiting closely similar fluorescent characters. A low-temperature technique proposed by Shpol’skii et al. [36] showed excellent band-narrowing features. In general, the combination of the low-temperature technique and SFS brings better resolution than the traditional fluorescence methods [37]. CESFS was extended to low-temperature condition by Inman and Winefordner [38], who analyzed PAHs in multicomponent samples with the proposed method. Shpol’ skii low-temperature technique was combined with NLVASFS to increase spectral resolution, which was demonstrated by the simultaneous identification and quantification of some PAHs in mixtures, as seen in Fig. 7. It was quite difficult to determine these PAHs in room-temperature. NLVASFS due to the broadbands of spectra [39].
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Chemometrics
Chemometrics, possessing the characteristics of resolving multicollinearity problems well, is one of the modern approaches to improve the selectivity of analytical methods. Chemometrics techniques are widely used in analytical chemistry, such
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Fig. 7 Contour plot of 1,2-benzoanthracene (1,2-BA, a), coronene (Cor, b), pyrylene (Pyr, c), 3,4-benzopyrene (BaP, d) at room temperature (a) and at low temperature (b), and the synchronous fluorescence spectra of the above PAHs mixtures at room temperature (a¢) and at low temperature (b¢). The bold folded lines are the selected nonlinear VASFS scan routes (Reproduced Ref. from [39], with permission from Elsevier)
as multivariate calibration, classification, pattern recognition, and clustering. Multivariate calibration approaches, especially, are often used for multicomponent analysis, such as principal components analysis, partial least squares (PLS), and parallel factor analysis. In the application of chemometric tools, generally, there are two sets of data, of which one is a set of calibration samples used to establish the mathematical models and the other is the unknown samples which is predicted by the mathematical models [40]. Multicomponent samples could be better investigated by using synchronous technique combined with a multivariate calibration method [41]. Berzas-Nevado et al. [42] combined PLS with nonlinear variableangle synchronous fluorimetry firstly for the simultaneous determination of multiple components in a spectrally overlapped mixture. CWSFS technique in combination with multiple linear regressions was used for the quantification of BaP in the presence of four PAHs [43]. Two multivariate techniques, principal component regression and partial least square regression coupled with CWSFS, have been successfully applied for the classification of petrol–kerosene mixtures. This method could be used for the estimation of kerosene in kerosene-mixed petrol [44].
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Rapid Determination of Polycyclic Aromatic Hydrocarbons in Foods
PAHs, consisting of fused aromatic rings, occur in the atmosphere, water, sediments, tobacco smoke, and food. Since some PAHs are highly carcinogenic and mutagenic, both the International Agency for Research on Cancer (IARC) and the United States Environmental Protection Agency (US EPA) suggest the 16 PAHs as priority pollutants [45]. Thus, it is of great significance to detect the PAHs in environment and food. The monitoring of PAHs in environmental samples, such as water, sediments, soil, and particulate air, has been widely studied [34, 46–48] while food samples have received much less attentions due to their complex matrices. BaP, one of the most carcinogenic and mutagenic PAHs, is recommended as an indicator of the presence of PAHs in diverse matrices. Both the IARC and the US EPA rank BaP as one of the most carcinogenic PAHs, and the Scientific Committee on Food (SCF) indicates that BaP can be used as a marker for the occurrence and impact of carcinogenic PAHs in food [49]. Therefore, there is no surprise that more and more concerns were paid to the rapid screening of BaP in food. There are various analytical methods reported for the determination of PAHs in several food matrices, and the common analytical techniques are GC/MS and HPLC-FLD. Due to the complexity of food samples, time-consuming and laborious cleanup processes are indispensable for chromatography-based techniques. Therefore, there is a critical need for rapid, simple, and economical techniques for the evaluation and monitoring PAHs in food. Because of the strong intrinsic fluorescence of PAHs, fluorimetry is especially suitable for the monitoring of PAHs. Synchronous fluorescence spectroscopy may be the most popular fluorescence technique for the simultaneous screening of PAHs. Micelle-sensitized CESFS was proposed for the simultaneous determination of PAHs mixtures free from organic solvents with the detection limits at a level of ppt [17]. CESFS and NLVASFS have shown to be potential tools for the rapid screening of BaP in fatty food and oil. Second-derivative technique and low-temperature technique have been introduced to further improve the selectivity and sensitivity.
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Rapid Screening of Benzo(a)pyrene in Foodstuff
Great efforts have been paid to the rapid screening of BaP in foodstuff, including fatty food, oil, tea, and so on. By choosing an appropriate energy difference for CESFS, it is possible to eliminate interferences and give the characteristic spectrum of BaP. The use of CESFS relieved the requirement for pretreatment of food samples, which was simply extracted by directly soaked in organic solvent [50] or by ultrasonic extraction. As shown in Fig. 8, CESF spectrum of a dried pork sample indicated a distinct peak of BaP which coincided with the spectral peak of standard BaP solution. By coupling CESFS with second-derivative technique, the selectivity and sensitivity of analysis were enhanced. As shown in Fig. 8a, the CESF spectrum of BaP was still interfered by the matrix. Second-derivative technique was used to provide
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better resolution and narrower peaks (Fig. 8b). Moreover, the derivative technique diminished the background interference of other fluorophores in the sample. Recently, dimethylsulfoxide (DMSO) is found to be an attractive solvent for the extraction of BaP in oil samples due to the high fluorescence intensity of aromatic hydrocarbons in DMSO and less interference. We suggested a novel method to extract BaP in oil samples by ultrasonication in DMSO. Second-derivative CESFS, with the advantages of the excellent band-narrowing and background-suppressing features, was introduced for the determination of the extracts, resulting in better selectivity and signal-to-noise ratio. The proposed method was validated by measuring BaP in a certified reference material (coconut oil BCR-458) and was compared with the national standards of the People’s Republic of China [51]. As shown in Fig. 9, the spectrum of the sample extracted by the proposed method was consistent with that extracted by national standards.
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Furthermore, in order to provide better resolution, low-temperature technique is introduced to CESFS for the analysis of PAHs in foods [52]. Figure 10 shows the comparison of the spectra obtained at room temperature and low temperature.
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Fig. 12 (a) Contour plots of BaP (solid lines), BkF (dash-dotted lines), and Ant (dashed lines); (b) derivative NLVASF spectra of BaP (1.0 mg/L, dash-dotted curve), BkF (3.0 mg/L, short dotted curve), Ant (10.0 mg/L, dashed curve), and the mixture with the same concentrations as above (solid curve) (Reproduced from Ref. [53], with permission from ACS)
As it can be seen, the spectral resolution of the compounds in food matrix in low temperature is much better than that in room temperature, making the quantification much easier and more accurate. The proposed method was evaluated by both the recovery experiments and the comparison of the spectra of samples with those of standard solutions, as shown in Fig. 11. Thus far, there are several researches focused on eliminating the interference of benzo(k)fuoranthene (BkF) to BaP in the fluorimetric detection, including the chemometrics techniques and chemical separation. NLVASFS has shown the potential to the rapid screening of BaP in tea samples among the serious interference of BkF and anthracene (Ant). Figure 12a showed the contour plots of BaP, BkF, and Ant. Obviously, the contour plot of BaP is overlapped by that of both BkF and Ant. By selecting an optimized scanning route, it is possible to gain spectral profiles of individual PAH in the PAHs mixtures and they make good consistency with the
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spectra of the single component, as shown in Fig. 12b. Good linearity is obtained with detection limits of 0.02, 0.08, and 0.14 mg/L for BaP, BkF, and Ant, respectively. The proposed method was successfully applied to the rapid screening of BaP, BkF, and Ant in tea samples extracted by ultrasonication in n-hexane [53].
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Rapid and Differential Analysis of Porphyrins in Human Body Samples
Porphyrins play very important roles in the metabolism process which is taking place in human body. The identification and quantitative determination of different porphyrins in body samples are essential for the understanding of mechanism and differential diagnosis of porphyrias and other porphyrin-related diseases, and also useful for monitoring the health-risk assessment of environmental pollutants. However, the porphyrins in body samples exhibit similar fluorescence spectra and overlap with each other seriously, which makes it difficult to determine the porphyrins simultaneously with conventional fluorescence spectroscopy. In order to improve the selectivity of spectrofluorometry, several novel spectrofluorimetric approaches have been developed for the analysis of metabolic prophyrins in human body samples. The fluorescence characteristics of various metabolic porphyrins, such as protoporphyrin, uroporphyrin, coproporphyrin, and zinc protoporphyrin, in different human body samples, including whole blood, serum, saliva, feces, and urine, were investigated. Derivative matrix isopotential synchronous fluorescence and nonlinear variable-angle synchronous fluorescence spectroscopic approaches were developed to resolve the spectral overlap of porphyrins in different body samples. PLS technique was also applied to increase the selectivity. By using suitable coupled techniques, the individual components of porphyrin multicomponent mixtures can be distinguished simultaneously and detected rapidly without resorting to the complicated separation procedures of these porphyrins. In this section, we summarize some simple analytical approaches for the differential identification and quantification of porphyrins in whole blood, serum, saliva, blood cells, feces, and urine. The proposed SFS-based approaches are rapid and cost-effective, which would provide efficient analytical tools for point-of-care tests, and especially for the rapid screening of differential porphyrins in a large number of body samples.
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The Spectral Discrimination of Coproporphyrin and Protoporphyrin in Feces and Saliva
Coproporphyrin (CP) and protoporphyrin (PP) in feces were simultaneously measured with first-derivative MISF technique [54]. Figure 13a shows the contour plots of CP and PP standard solutions, which are similar and overlapped seriously. It is difficult to resolve the CP and PP by direct, conventional SFS while first-derivative MISFS provides a simple approach to detect these porphyrins simultaneously.
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Fig. 13 (a) Theoretical contour maps of 175 nmol/L CP (thin solid lines) and 500 nmol/L PP (dashed lines), and the determination route (thick solid line). Trajectory i and ii are the detection routes of CP and PP, respectively. (b) First-derivative MISF spectra of 110 nmol/L CP (dotted curve), 250 nmol/L PP (dashed curve), and their mixture (solid curve) (Reproduced from Ref. [54])
A suitable scanning route is essential for MISF measurements. The scanning route was chosen according to the fluorescence information from the three-dimensional spectra of CP and PP. In Fig. 13a, Trajectory i and ii are the detection routes of CP and PP, respectively. The two contour lines were united as an integrated detection route with a crossing point. Using the above optimized trajectory, MISF spectra of CP and PP in standard solutions were recorded, and they resolved well with almost no mutual interference (Fig. 13b). The analytical selectivity is enhanced by using first-derivative technique. The amplitudes of the positive- and negative-derivative peaks were measured for the quantification of these two porphyrins. A fecal specimen from a pregnant woman was analyzed by the absorbance, fluorescence excitation, and MISF techniques. As seen in Fig. 14, spectrophotometry produced the least distinct spectrum, which was not good for analytical purposes. MISF spectrometry produced a well-structured spectrum and made it possible to identify and quantify coproporphyrin and protoporphyrin simultaneously. Furthermore, derivative MISFS was applied to develop a rapid and simple approach for the simultaneous detection of CP and PP in saliva, the matrix of which is quite different from that of feces. The MISF scanning route was optimized carefully according to the contour plots of CP and PP in saliva. The proposed method offered good resolution of CP and PP in saliva, with detection limits of 0.87 and 0.46 nmol/L, respectively.
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Simultaneous Analysis of Coproporphyrin and Uroporphyrin in Urine
Coproporphyrin and uroporphyrin (UP), possessing the close similar structure, exhibit seriously overlapped fluorescence spectra (Fig. 15a), and hence they could
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Fig. 14 Absorbance (a), fluorescence excitation (lem = 668 nm; b), and MISF (c) spectra of a fecal sample from a pregnant woman containing 10.3 nmol of CP and 27.0 nmol of PP per gram dry weight (Reproduced from Ref. [54])
b
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UP 660 640 CP 620 ii 600 580 560
i 380
390 400 410 Excitation Wavelength (nm)
420
Derivative Fluorescence Intensity
a
250 200 150 100 50 0 -50 –100 –150 –200 –250
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CP 0
100 200 300 400 Determination Series
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Fig. 15 (a) Contour maps of CP (solid lines) and UP (dashed lines) at a concentration of 112 nmol/L each, and the selected MISF scanning route (thick solid lines). Trajectory i and ii are the detection routes of CP and UP, respectively. (b) Derivative MISF spectra of 60 nmol/L CP (dotted curve), 52 nmol/L UP (dashed curve), and their mixture (solid curve) (Reproduced from Ref. [55], with permission from Elsevier)
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not be simultaneously determined by the conventional fluorescence techniques, even by CWSFS, where it is evident that a mixture of CP and UP exhibits only one merged peak. Derivative MISFS is an attractive alternative to solve this problem with simple extraction of CP and UP from urine samples. The careful examination of the contour plots of CP and UP leads to the selection of an optimum isopotential scanning route, indicated by Fig. 15a. Scanning along the selected isopotential trajectory, it is easy to obtain the synchronous fluorescence spectra of CP and PP, as shown in Fig. 15b. It is clear that the spectral bands of CP and UP are resolved satisfactorily. By combining MISFS with the derivative technique, it became possible to determine these two components separately with high selectivity and sensitivity [55].
5.3
Simultaneous Determination of Porphyrins in Blood Samples
Some simple and low-cost analytical techniques have been established, such as derivative VASFS, for the simultaneous determination of protoporphyrin IX and zinc protoporphyrin IX in whole blood samples [56], derivative, matrix-isopotential, NLVASFS (DMI-NLVASFS) for the rapid simultaneous determination of protoporphyrin IX, coproporphyrin III, and zinc protoporphyrin IX in ultramicro amount of erythrocytes, and nonlinear variable-angle synchronous fluorescence technique coupled with PLS regression for the rapid screening of protoporphyrin IX, uroporphyrin III, and coproporphyrin III in human whole blood. Besides, a new solvent, N, N-dimethylformamide, is used to extract porphyrins in blood [57]. The spectra of protoporphyrin IX and zinc protoporphyrin IX (ZnPP) overlapped seriously, and it was impossible to determine them by conventional SFS due to their seriously overlapped CWSF spectra. In order to simultaneously determine PP and ZnPP in whole blood samples, derivative VASFS is proposed by carefully choosing the scanning path. Figure 16 shows the contour maps of PP and ZnPP, and the VASF scanning path. It is obvious that the primary peak of PP was interfered by the secondary peak of ZnPP; thus, the point (lex = 385 nm and lem = 632 nm) was chosen as the detection point for PP, but not the fluorescence maximum point (lex = 406 nm and lem = 632 nm). The fluorescence maximum point of ZnPP (lex = 419 nm and lem = 590 nm) was selected as the detection point for ZnPP. Other points were chosen to make up a whole scanning route. With the selected scanning route for derivative VASF, PP and ZnPP were distinguished from each other simultaneously and rapidly. The spectra were resolved well, and the two components were determined in a single scan. It is also possible to make use of derivative MISFS to distinguish these two porphyrins in blood. We established derivative MISF technique for the rapid and simultaneous quantification of ZnPP and PP extracted from whole blood samples [58]. Compared with the derivative VASF technique, derivative MISF method has a better resolution while maintaining the strongest fluorescence intensity of PP due to the selected scanning route passing through the fluorescence maximum point of PP (Fig. 17).
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Emission Wavelength (nm)
700 680 660 640 620 600 580
ZnPP 560 320
340
360
380
400
420
440
460
Excitation Wavelength (nm)
Fig. 16 Contour maps of PP (thin solid curve) and ZnPP (dash-dotted curve), and the VASF scanning path (thick solid lines) (Reproduced from Ref. [56])
PP
Derivetive Fluorescence Intensity
300 200 100 0 −100 −200
ZnPP
−300 −400 −500 0
200
400 600 Determination Series
800
1000
Fig. 17 Derivative MISF spectra of PP (dashed curve), ZnPP (dash-dotted curve), and the mixture with the same concentration as before (solid curve) and solvent (dotted curve) (Reproduced from Ref. [58])
It is clear that the researches mentioned above are based on SFS to resolute porphyrins in human body samples, with the advantages of characteristics of spectral simplification, light scattering reduction, and selectivity improvement. Another approach is chemometric methods with the characteristics of resolving multicollinearity problems well. Furthermore, the combination of synchronous fluorescence techniques with chemometric methods could provide better performances. Partial least squares (PLS) regression in combination with nonlinear variable-angle synchronous fluorescence technique is proposed to solve the spectral overlapping problem of protoporphyrin IX, coproporphyrin III, and uroporphyrin III while the fluorescence intensity of PP, CP, and UP is maintained at the highest. The proposed method has been successfully applied to the blood samples and satisfied results were
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Table 1 Recoveries in blood samples by nonlinear VASFS coupled (Reproduced from Ref. [59], with permission from Elsevier) Added (nmol/L) Predicted (nmol/L) Samples PP UP CP PP UP CP 1 50 – 10 44.3 0.4 9.3 2 12.5 10 10 11.2 8.7 8.5 3 20 3.5 5 17.5 3.2 4.4 4 30 15 10 24.1 13.6 9.3
Recovery (%) PP UP 88.6 – 89.6 87 87.5 91.4 80.3 90.5
CP 93 85 88 93
700 675 650
PP B
625 600
A
CP
575 325
C D E
ZnPP 350
375
400
425
Excitation Wavelength (nm)
450
Derivative Fluorescence Intensity
b
a Emission Wavelength (nm)
with PLS regression
900 600 300
PP
0 –300
CP
–600
ZnPP –900
0
200
400
600
800
1000 1200
Determination series
Fig. 18 (a) Theoretical contour maps of 33 nmol/L PP (solid lines), 14 nmol/L CP (dashed lines), and 18 nmol/L ZnPP (short dotted lines). The scanning path ABCDE is for DMI-NLVASF spectrometry. (b) DMI-NLVASF spectra of the extract of erythrocytes (solid curve), spiked erythrocytes (dashed curve), which were spiked with 6.6 nmol/L PP, 5.5 nmol/L CP, and 6.4 nmol/L ZnPP, and the standard mixture of 13 nmol/L PP, 11 nmol/L CP, and 12.8 nmol/L ZnPP (dash-dotted curve) (Reproduced from Ref. [60])
obtained, with the recoveries ranged from 80.3 to 91.4% (Table 1). Five identically spiked blood samples are analyzed with the relative standard deviations less than 5%, respectively [59]. For the analysis of PP, CP and ZnPP in erythrocytes, it is hard to simultaneously determine these porphyrins by MISFS or VASFS due to the serious overlap of spectra, as indicated in Fig. 18. Hence, we combine these two synchronous techniques and the first-derivative technique, named DMI-NLVASFS to accomplish the goal. This approach required only ultramicro amount of erythrocytes since it was very sensitive. By using the similar manner as the above studies, a scanning route was carefully selected for the DMI-NLVASF detection. It was easy to get high resolution of CP and PP via scanning the MISF route A–B–C. By coupling with VASF route C–D–E, it was possible to determine the PP, CP, and ZnPP simultaneously. This method was successfully applied to the analysis of PP, CP, and ZnPP in ultramicro erythrocytes. As seen in Fig. 18b, the peak positions of each porphyrin in the spectra of extract of erythrocytes, spiked erythrocytes, and the mixed standard solution of porphyrins were the same. The background matrix of the extract of blood cells and spiked blood cells was eliminated by the derivative technique [60].
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Conclusions
SFS, providing simple spectra, narrow bandwidth, better resolution, and flexibility, becomes one of the attractive alternatives for the simultaneous analysis of multicomponent samples with seriously overlapping conventional spectra. Generally, the SFS-based approaches possess the quality of being sensitive, selective, simple, rapid, and cost-effective. By using these novel approaches, it is possible to make sample pretreatments simple and time saving. The combination of SFS approaches and other techniques, such as derivative technique, low-temperature technique, and chemometrics, has improved its performances in practical application. These techniques provide potential tools for clinical analysis and food safety evaluation. Acknowledgment We are grateful for the financial support from the National Natural Science Foundation of China (29875023, 20575055, and 20975084), the National Basic Research Program of China (973 Program, 2007CB935600), the Educational Ministry Foundation of China, the Natural Science Foundation of Fujian Province (No. B0410002), the Science and Technology Program of Fujian Province (2009Y0046), and the Science and Technology Project of Xiamen. We thank Yu-Luan Chen for critical reading of this manuscript.
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