Food Chemistry 159 (2014) 309–315

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Analytical Methods

Selective identification and quantification of saccharin by liquid chromatography and fluorescence detection Sergio N.F. Bruno a,⇑, Carlos R. Cardoso a, Márcia Mosca A. Maciel a, Lidmila Vokac a,b, Ademário I. da Silva Junior b,⇑ a b

Advanced Laboratory Service – SLAV-RJ/LANAGRO-MG/MAPA, Av. Maracanã, 252, CEP 20271-110 Rio de Janeiro-RJ, Brazil Instituto Federal do Rio de Janeiro (IFRJ), Campus Rio de Janeiro, Av. Senador Furtado, 121, CEP 20270-021 Rio de Janeiro-RJ, Brazil

a r t i c l e

i n f o

Article history: Received 14 January 2013 Received in revised form 8 October 2013 Accepted 1 March 2014 Available online 14 March 2014 Keywords: Selective saccharin determination Saccharin fraud Fluorescence detection in liquid chromatography

a b s t r a c t High-pressure liquid chromatography with ultra-violet detection (HPLC–UV) is one of the most commonly used methods to identify and quantify saccharin in non-alcoholic beverages. However, due to the wide variety of interfering UV spectra in saccharin-containing beverage matrices, the same method cannot be used to measure this analyte accurately. We have developed a new, highly effective method to identify and quantify saccharin using HPLC with fluorescence detection (HPLC–FLD). The excitation wavelength (250 nm) and emission wavelength (440 nm) chosen increased selectivity for all matrices and ensured few changes were required in the mobile phase or other parameters. The presence of saccharin in non-diet beverages – a fraud commonly used to replace more expensive sucrose – was confirmed by comparing coincident peaks as well as the emission spectra of standards and samples. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Saccharin (o-benzoic sulfimide, C6H4COSO2NH) was discovered accidentally by Fahlberg and Remsen (1879) while they were studying the oxidation of o-toluene-sulfonamides. In 1984, the acceptable daily intake (ADI) for saccharin was established as 2.5 mg/kg of body weight (Assumpção, Medeiros, Madi, & Fatibello-Filho, 2008). Later, the ADI for saccharin (expressed as saccharin acid) was modified to 5 mg/kg of body weight (Food Standards Agency, 2002). In 1988, the Brazilian Ministry of Health Resolution number 04 established a maximum limit of 30 mg/100 mL for this sweetener in products sold in Brazil (Brasil, 1988). Saccharin is about 300 times sweeter than sucrose, but it has a bitter and metallic aftertaste (Assumpção et al., 2008; Glória, 2000). A mixture of saccharin and sodium cyclamate (1:10) produces the desired sweetness (Glória, 2000) since the bitter aftertaste of saccharin is masked by cyclamate and the unpleasant aftertaste of cyclamate by the saccharin. Also, the sweetening effect of this mixture is enhanced by a synergism between the ingredients (Zygler, Wasik, & Namies´nik, 2009).

⇑ Corresponding authors. Tel.: +55 21 2710 7431; fax: +55 21 2569 1198 (S.N.F. Bruno). E-mail address: [email protected] (S.N.F. Bruno). http://dx.doi.org/10.1016/j.foodchem.2014.03.001 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Assays for the identification of saccharin using reactions that develop color are common and have been described widely (FSSA, 2012; Wood, Foster, Damant, & Key, 2005). Identification of saccharin using HPLC–UV is based on retention time, either by comparing sample retention time with appropriate standards or by simultaneously injecting the standard with test solutions (Food Standards Agency, 2002; Wood et al., 2005). The methods available for saccharin determination in food include UV–VIS spectroscopy, differential pulse polarography, gravimetric analysis, fluorimetry, sublimation, potentiometry, micellar electrokinetic capillary chromatography and HPLC (Fatibello-Filho, Vieira, Gouveia, Calafatti, & Guaritá-Santos, 1996; Food Standards Agency, 2002; Zygler et al., 2009). Until recently, only one spectrophotometric method and two HPLC–UV methods had been included in European Standards (Institute for Reference materials, 2010; Wood et al., 2005). In 1975, Nakamura (apud Fatibello-Filho et al., 1996) described a fluorimetric method for saccharin with excitation wavelength at 277 nm and emission wavelength at 410 nm. However, this method was slow with many steps and required heating. Since spectrophotometric, gravimetric and electrochemical methods are very often too time consuming, HPLC in combination with ultraviolet (UV) detection has become the method of choice to determine saccharin in international regulations (Agilent Technologies, 2001; Glória, 2000; Zygler et al., 2009). Moreover, HPLC has high selectivity (Assumpção et al., 2008; Food

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Standards Agency, 2002; Wasik, McCourt, & Buchgraber, 2007), and CODEX suggests UV detection at 227 nm and a C-18 reversed phase column to determine saccharin, acesulfame-K, saccharin and benzoic and sorbic acids in beverages and jams, simultaneously (Food Standards Agency, 2002; Hannisdal, 1992). In 2004, this CODEX considered stopping saccharin control in beverages and sweets because of the absence of measures of uncertainty meaning usage levels for saccharin and other sweeteners would exist only for chocolate and cocoa products (Codex Committee on Methods of Analysis & Joint FAO/WHO Food Standards Programme, 2004). However, since saccharin has the best cost to sweeteningpower ratio and is cheaper than sucrose (Porto, 2010); the Brazilian food industry has continued to replace sugar with saccharin in non-diet products, as shown in this work. The use of high performance liquid chromatography with evaporative light scattering detection (HPLC–ELSD) for saccharin identification and detection is more recent (Wasik & Buchgraber, 2007; Wasik et al., 2007) as well as HPLC–MS (Yang & Chen, 2009; Zygler, Wasik, Kot-Wasik, & Namies´nik, 2011). Ujiie, Hasebe, Chiba, and Yanagita (2007) used HPLC/MS/MS for the simultaneous determination and identification of seven kinds of food preservatives and saccharin in food. Xian-Ming and Hong-Mei, (2007) introduced a new method for sodium saccharin in food using HPLC–FLD with 97% recovery and a linear concentration range from 0.005 to 0.20 mg/mL. They suggested their method eliminated the interference observed in wine and chocolate samples using HPLC–UV. However, since it was the first trial published using FLD for saccharin detection, some gaps remained. For example, they used only one emission wavelength (420 nm), and did not investigate any options for better selectivity or unambiguous identification. Consequently, the method was not validated. Within the wide variety of non-alcoholic beverages containing saccharin, many components – such as certain flavorings and tannins – elute at similar times to saccharin and absorb at the same selected wavelengths (214–217, 227–228, 254, 265 nm), resulting in poor selectivity. Various combinations of columns, chemical modifiers and wavelengths have been used to achieve the appropriate separation and eliminate interfering species (Food Standards Agency, 2002; Glória, 2000; Zygler et al., 2009). However, most of these methods usually refer to, and are limited to a number of matrices such as soft drinks, instant powder drinks, etc. (Glória, 2000; Wood et al., 2005; Zygler et al., 2009). When the matrix changes, such as in mate tea and currant syrup – see Section 3.4.2 – adjustments to the chemical modifier, gradient elution or even to the column characteristics rarely achieve acceptable resolution. Therefore, these HPLC–UV methods are not suitable for the analysis of a broad range of products or matrices samples unless corrections are made and the methods are adapted to the new conditions. However, making these changes is as tedious and time consuming as the alternative methods mentioned previously. Therefore, the purpose of this study was to define a highly selective validated method HPLC–FLD method, which could unambiguously quantify saccharin in a broad range non-alcoholic beverages. This would facilitate identification of frauds arising from the addition of saccharin in standard (non-diet), non-alcoholic beverages and also control of saccharin levels in diet beverages. The method described requires almost no modification to the mobile phase or any other parameter that has not been previously defined. A worldwide inter-laboratory study involving 57 organizations participated in a Food Analysis Performance Assessment Scheme (FAPAS, 2009) to validate the method. Whenever possible, saccharin quantification was also performed with DAD (diode array detector) to compare spectra and retention time.

2. Experimental 2.1. Apparatus and experimental conditions All standard solutions and samples were injected under isocratic conditions into two Agilent 1200 Series liquid chromatographs. Each chromatograph was equipped with a Quaternary Pump G1311A, Rheodyne 7725i Injector (20 lL loop), Fluorescence Detector G13A1, Diode Array Detector G1315D, Column Thermostatic Chamber at 25 °C, and Vacuum Degasser G1316A. The columns installed were SGE SS Exsil ODS (250  4.6 mm ID  5 lm) (column 1) – with Exsil ODS2 (10 mm  4 mm ID  5 lm as precolumn – LiChrosorb RP18 (250  4.6 mm I.D.  10 l) (column 2), and a Synergi-Phenomenex Polar-RP (150  4.6 mm  4 l) (column 3) with Zorbax Stablebond SB C-18 DE 4.6  12.5 mm  5 lm as pre-column. Chromatographic runs were performed in methanol 15% (v/v) and acetic acid 1 mol L 1, adjusted with NaOH 50% to pH = 3.2. Flow values were from 0.6 to 1.0 mL min 1. Excitation wavelength was 250 nm and emission wavelengths for fluorescence detection were 440 and 550 nm. Analytical parameters were controlled by Agilent G2170BA software. The method was modified to methanol 10% for the two most difficult matrices. In column 3, all DAD experiments were conducted with H3PO4 0.1% and methanol 1:1 (v/v) as mobile phase and main wavelength to quantification at 220 nm. 2.2. Reagents All solutions were prepared with analytical grade chemicals and water from a Milli-Q system. The 0.1 mol L 1 saccharin stock solutions were prepared from their sodium salt, stored under refrigeration, and used within a month time period. The working solutions were prepared from this stock solution and used only for the trials conducted in the same day. 2.3. Analytical procedure Qualitative validation parameters and quantitative parameters (such as precision, selectivity, robustness, linearity, LOD and LOQ) were selected in accordance to Brazilian law (Albano & Raya-Rodriguez, 2009; Inmetro DOQ-CGCRE-008, 2010). 2.3.1. Identification Saccharin retention times (tR) for sample and standards were measured at 440 nm (emission). If the tR values were coincident, the sample emission spectra were compared to reference emission spectrum using software. If the emission spectrum coincidence was 98–99% or higher, peak areas at emission wavelengths 440 and 500 nm were calculated and the area at 440 nm was divided by the area at 500 nm. The presence of saccharin in a sample was confirmed if the product value was within 2.6–3.0. This range derived from the calibration curves determined during the identification and quantification trials. When the analyte concentrations were close to the limit of quantification, a larger volume was injected (e.g., 100 lL) to promote effective coincidence. In more complex samples, the presence of saccharin was also confirmed through the absorbance peak retention time coincidence at 220 nm and the emission peak at 440 nm as well as comparing the sample absorbance spectrum with the reference absorbance spectrum. 2.3.2. Determination An external standard curve was obtained through the linear regression of emission peak area (y) at 440 nm and saccharin concentration in mg/100 mL (x). In the presence of saccharin, the

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emission peak area can be used to calculate saccharin concentration (FSSAI, 2012; Xian-Ming & Hong-Mei, 2007).

will be validated in the future, as it increases the range of the method.

2.3.3. Sample collection and treatment before injection Samples were collected from producers or purchased in the market and taken to our laboratory (SLAV-RJ/LANAGRO-MG) by the Serviço de Fiscalização de Produtos de Origem Vegetal/RJ-MAPA (RJ-MAPA, Surveillance Service for Vegetable Origin Products). Fruit juices, soda drinks and other refreshments were not diluted prior to analysis unless saccharin concentrations were anticipated to exceed the linear range, particularly for samples that had the sweetener concentration indicated in the labels. Carbonated drinks were subjected to ultrasound (5 min) to eliminate CO2. Syrups, concentrated fruit juices and liquid and solid concentrates were diluted as recommended by suppliers and then sampled. Again, those beyond the linear range were diluted further. Before injection, all samples were filtered through high volume membrane filters 13 mm  0.45 lm (Durapore, in PVDF).

3.2. Identification essays

3. Results and discussion 3.1. Optimization of analytical parameters Method elaboration started with column 1 and UV (DAD) as the detector. Column 2 was used with the operational conditions described in literature (Food Standards Agency, 2002; FSSAI, 2012; Glória, 2000) and evolved to fluorescence detection, after testing wavelength, mobile phase composition and elution gradient to avoid interference (Agilent, 2006); adequate separation in different kinds of drinks was achieved using this approach. During these trials, a clearly resolved photoemission peak for saccharin emerged, and the excitation and emission wavelengths were selected for the best output. Column 3 enabled saccharin to be detected in several drinks, even with DAD. However, only fluorescence selectively detected saccharin in most of the drinks tested in column 1 and 2 (see Section 3.4.2). The only samples where interference at the same wavelength occurred were grape juice and mate tea. A slight modification in the mobile phase – to 90% from 85% water – was enough to correct this (Fig. 3c (I and II) and d). This new condition

The ratio between peak areas at 440 and 500 nm was selected because it was almost free from interferences and had higher responsiveness. This ratio is shown in Table 1 for distinct saccharin concentrations in samples and standards. Overall, 164 trials were conducted that covered 10 standard (non-diet) products and two diet products. The presence of saccharin was detected in 22% of these samples. About 45% of standard syrups (non-diet) and 20% of standard products (non-diet) contained saccharin (see Table 2), which should not be present under international regulations. 3.3. Examples of quantified samples The saccharin quantification results for 10 samples that should contain it and those from the most complex matrices are presented in Table 3. Overall, the results for products supposed to contain saccharin are in agreement with producers’ declaration. 3.4. Performance and acceptance criteria 3.4.1. Linearity Five solutions containing different analyte concentrations within the working range were prepared and each one was injected three times. The resulting analytical curve can be seen in Fig. 1. Cochran test and the randomness of calibration residues confirmed the homoscedasticity of this analytical curve (detail in Fig. 1). 3.4.2. Selectivity Samples with and without saccharin from distinct matrices and within the working range (Xian-Ming & Hong-Mei, 2007) were injected in triplicate in the equipment. The high resolution obtained by HPLC–FLD method (Rs P 2.76 for most of the matrices) is well beyond the minimum value (Rs P 1.50) required by international legislation (OIV, 2012). The absence of saccharin retention time

Fig. 1. Saccharin calibration curve obtained by HPLC–FLD in column 2. Residues plot in detail.

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Fig. 2. (a) Chromatogram of guaraná with saccharin (FLD) using column 1; tR (saccharin) = 6.393 min and flow = 1.0 mL min 1. (b) Chromatogram of Blackcurrant syrup sample obtained by HPLC–UV (220 nm, column 2); saccharin’s tR = 3.694 min and flow = 0.6 mL min 1. (c) Chromatogram of Blackcurrant syrup sample obtained by HPLC–UV (220 nm, column 3); tR saccharin = 2.767 min and flow = 1.0 mL min 1. (d) Chromatogram of Blackcurrant syrup sample obtained by HPLC–FLD – emission at 440 nm (column 2) – that shows high similarity with saccharin standard (99%, 98%); tR saccharin = 3.491 min; flow = 0.6 mL min 1. (e) Chromatogram of Mate tea with lemon (HPLC–UV/ 220 nm) in column 2; tR interfering species = 3.238 min; flow = 0.8 mL min 1.

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Fig. 3. (a) Lack of similarity between absorption UV-spectra of unknown compound refered in Fig. 2(e) with saccharin similar retention time and saccharin reference spectrum (in red). Similarity is 9.4%. (b) Chromatogram of Mate tea with lemon (HPLC–FLD; emission at 440 nm) in column 2 and flow = 0.6 mL min 1 with its unknown compound with saccharin similar retention time in the detail. Emission spectral similarity of only 60.8%. Saccharin reference spectrum in red in the detail. (c) Diet grape juice determination. (I) Chromatogram with 15% (v/v) MeOH as mobile phase. (II) Chromatogram with 10% (v/v) MeOH as mobile phase. Both with 0.6 mL min 1. (d) Mate tea chromatogram with saccharin addition. Chromatogram with 10% (v/v) MeOH as mobile phase in column 2 and flow = 0.6 mL min.

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Table 1 Ratio between areas at 440 and 500 nm emission wavelengths for standard solutions and samples with saccharin (n = 3). Maximum estimated error for area ratio was 0.02. Saccharin (mg/100 mL)

Average area (440 nm)

Average area (500 nm)

Area ratio (440/500 nm)

7.27 14.54 23.87 29.06 36.33 Solid mixture for passion fruit drink Solid mixture for passion fruit drink Solid mixture for guava drink

192.1 377.2 608.6 730.4 905.3 218.4 104.1 122.2

65.1 129.3 213.7 258.2 323.4 73.4 34.2 40.9

2.95 2.92 2.85 2.83 2.80 2.98 3.04 2.98

Table 2 Presence of saccharin in non-alcoholic beverages (diet and non diet). Matrix

Syrup Soft drink Refreshment Juice Nectar Mixed beverage Solid mixture Liquid mixture Diet iced tea Diet liquid mixture Coconut water Liquid concentrate Total

Number of samples

47 44 22 15 7 5 7 1 1 1 1 11 162

Results Positive

%

21 2 1 0 0 0 3 1 1 1 0 6 36

44.7 4.5 4.5 0.0 0.0 0.0 42.9 0.0 100.0 100.0 0.0 54.5 22.2

Table 3 Saccharin content in samples of non alcoholic beverages overall considered to be the most complex to determine. Results ± standard deviation (n = 3); excitation at 250 nm and emission at 440 nm. Sample

Concentration (mg/100 mL)

Guarana syrup Guarana syrup Mixed syrup guarana/açai Blackcurrant syrup Grape syrup Guarana syrup Mixed Refreshment guarana/açai Guarana soda Blackcurrant soda Guarana soda Blackcurrant syrup Low calorie mate Mixed syrup guarana/ginseng Blackcurrant syrup Light grape juice

53.1 ± 0.53 42.2 ± 0.21 65 ± 0.07 72.2 ± 0.47 67.5 ± 0.45 63.2 ± 2.41 2.1 ± 0.02 6.4 ± 0.12 4.2 ± 0.01 1.0 ± 0.03 17.4 ± 0.02 9.9 ± 0.17 15.5 ± 0.12 19.1 ± 1.22 2.8 ± 0.02

peaks (tr = 6.4 min) was confirmed through the use of fluorescence detector (excitation at 250 nm and emission at 440 nm) when necessary. Fig. 2(a) shows a chromatogram using column 1 for a guaraná beverage sample with saccharin. Fig. 2(a–e) show interference as a result of the matrices using HPLC–UV – as can be seen in blackcurrant syrup (Fig. 2b and c) and mate tea (Fig. 2e), which was eliminated using HPLC–FLD (Fig. 2a and d). Fig. 2(b and c) also show that, when blackcurrant syrup samples were analyzed using HPLC–UV, saccharin was poorly separated with column 2 and well separated using column 3. Fig. 2(d) shows that FLD (k = 440 nm) enabled saccharin identification and quantification in blackcurrant syrups using column 2. The peak’s high purity at retention time of 3.291 min shown in the top of the figure (similarity greater than 99% with the peak from

the spectra library) indicated the presence of saccharin and the absence of interference. The other emission band at 500 nm was also detected without interference. Since the ratio between these band areas is close to 2.73, this confirms the presence of saccharin where it should not be and characterizes the fraudulent use of a sweetener rather than sugar in a standard (non-diet) beverage. The chromatogram from non-diet mate tea with lemon in Fig. 2(e) shows a sample component that has a retention time similar to that of saccharin in HPLC–DAD. However, using DAD, this peak did not resolve, even with the most efficient column 3. However, because DAD captures whole absorption spectrum, Fig. 3(a) shows the unknown component is not saccharin, since the spectrum is different. Fig. 3(b) shows mate tea with lemon analyzed by HPLC–FLD (emission at 440 nm). Column 2 delivered a better separation for the unknown compound with retention time similar to saccharin, even though some tailing was observed. However, the lack of spectrum similarity persisted and the area ratio, between emission peaks at 440 and 500 nm, was out of the range (1.9), which confirmed the absence of saccharin. Figs. 2 and 3 also substantiate the improved selectivity of the proposed method. Thus, the high selectivity of this method can be linked to the excellent identification – as described in Section 2.5 and 3.1 – making FLD detection suited for saccharin determination, as shown in Fig. 3(c I–II, and d). The emission spectra from interfering species show that they are distinct substances – probably phenolic compounds (Perumalla & Hettiarachchy, 2011). 3.4.3. Precision Coefficients of variation were used to assess precision. Repeatability (same analyst) was measured at three levels (50, 100 and 150 mg/100 mL) using a guaraná sample without saccharin. Six repeated measurements were performed and the coefficient of variation (CV) was calculated for each concentration. The Horwitz limit was also calculated for each level, as the maximum CV limit (Horwitz & Albert, 2006). The maximum experimental CV was 0.5% and the minimum Horwitz limit was 6.49. All results were within acceptable limits. The evaluation of intermediate precision was performed at three saccharin concentration (50%, 100% and 150% of maximum limit) using the same guaraná sample. Twice two analysts repeated six measurements for each concentration on different days using different chromatographic systems, and the average CV was calculated for each level. The maximum CV was 0.72%. 3.4.4. Accuracy Determination of saccharin in a certified soft drink sample (cola soft drink) was used to assess method accuracy in a Food Analysis Performance Assessment Scheme, FAPAS. This resulted in an acceptable value (18.3 mg L 1 compared to 16.20 mg L 1, Z-score 1.23) from 57 laboratories worldwide (FAPAS, 2009). A guaraná sample containing 2.25 ± 0.03 mg L 1 was used to determine recovery with added (saccharin) concentrations 4.05, 12.15 and

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24.3 mg/100 mL. Recovery values of 101.4%, 98.6% and 98.5%, respectively, demonstrated the accuracy of the method. 3.4.5. Detection limits (LOD) and method quantification (LOQ) A guaraná sample containing 3 mg/100 mL of the analyte was diluted until the peak purity showed a significant decrease (from 99% to about 90% of similarity). After a 10-fold dilution, only the retention time could correlate the signal obtained for the blank sample with the analyte (saccharin). The LOD, 0.44 mg/100 mL, was calculated according to the equation ‘‘average for blank with analyte (X) + tS’’, where S is the standard deviation of seven measures and t, the Student’s t-distribution for a 99% interval reliability (six degrees of freedom). The LQM, 0.55 mg/100 mL, was calculated using the equation using the equation ‘‘average for blank with analyte + 10  S’’ (99% reliability). 3.4.6. Robustness Six assays of a 1.0 mg/100 mL standard at four pH values and temperatures were tested to determine the robustness of the method. As the robustness standard deviation (0.079) was lower than the reproducibility standard deviation (0.090), this method can be considered robust. 4. Conclusions This new method (HPLC–FLD) identified saccharin and determined its concentration in several soft drinks. It was efficient, selective, fast and precise, and showed an appropriate sensitivity to the target analyte. Proficiency and recovery tests indicated the accuracy of the method. Its application in an inter-laboratory trial was also successful. In the few of the matrices, where certified conditions were not appropriate, slight modification of the proposed method resolved the chromatograph sufficiently for quantification. Acknowledgements We would like to thank the LANAGRO-MG Coordination Office and its Laboratory General Coordination (CGAL) for their support during the research work developed at SLAV-RJ. This work initiates the collaboration between SLAV-RJ and IFRJ, Rio de Janeiro campus, through the cooperation with a researcher from its NCQ group. We also thank IFRJ for its support. References Agilent 1200 Series Fluorescence Detector G1321a (2006). User Manual. Edition 02/06. URL: ; Accessed 08.16.12. Agilent Technologies (2001). HPLC for Food Analysis. URL: . Accessed on 08.10.12. . Albano, F. M., & Raya-Rodriguez, M. T. (2009). Validação e Garantia da Qualidade de Ensaios Laboratoriais. Rede Metrológica: Porto Alegre – RS, Brazil. 1st ed. Assumpção, M. H. M. T., Medeiros, R. A., Madi, A., & Fatibello-Filho, O. (2008). Development of a biamperometric procedure for the determination of saccharin in dietary products. Quimica Nova, 31, 1743–1746.

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Selective identification and quantification of saccharin by liquid chromatography and fluorescence detection.

High-pressure liquid chromatography with ultra-violet detection (HPLC-UV) is one of the most commonly used methods to identify and quantify saccharin ...
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