a novel cation exchange polymer as a reversed-dispersive solid phase extraction sorbent for the...
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Title: A novel cation exchange polymer as areversed-dispersive solid phase extraction sorbent for the rapiddetermination of rhodamine B residue in chili powder andchili oil
Author: Dawei Chen Yunfeng Zhao Hong Miao Yongning Wu
PII: S0021-9673(14)01868-8DOI: http://dx.doi.org/doi:10.1016/j.chroma.2014.11.070Reference: CHROMA 356056
To appear in: Journal of Chromatography A
Received date: 10-7-2014Revised date: 13-10-2014Accepted date: 27-11-2014
Please cite this article as: D. Chen, Y. Zhao, H. Miao, Y. Wu, A novel cationexchange polymer as a reversed-dispersive solid phase extraction sorbent for therapid determination of rhodamine B residue in chili powder and chili oil, Journal ofChromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.070
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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A novel cation exchange polymer as a reversed-dispersive solid 1
phase extraction sorbent for the rapid determination of rhodamine B 2
residue in chili powder and chili oil 3
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Dawei Chen, Yunfeng Zhao, Hong Miao*, Yongning Wu 5
6
Key Laboratory of Food Safety Risk Assessment, Ministry of Health; China National 7
Center for Food Safety Risk Assessment, Beijing 100021, China 8
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*Correspondence: Hong Miao 10
E-mail: [email protected] 11
Tel: +86-10-67770158 12
Fax: +86-10-67790051 13
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Abstract 26
This paper presents a new analytical method for the determination of rhodamine B 27
(RB) residue in chili powder and chili oil based on a novel reversed-dispersive solid 28
phase extraction (r-dSPE) and ultra high performance liquid chromatography-high 29
resolution mass spectrometry (UHPLC–HRMS). Chili powder and chili oil samples 30
were first extracted with acetonitrile/water (1:1 v/v) and acetonitrile, respectively. 31
Then, RB from the extract was adsorbed to the polymer cation exchange (PCX) 32
sorbent with the characteristics of ion exchange and reversed-phase retention. 33
Subsequently, the analyte in PCX sorbent was eluted with ammonium 34
hydroxide/methanol (1:99 v/v) through a simple unit device equipped with 1 mL 35
syringe and 0.22 μm nylon syringe filter. All of the samples were analyzed by 36
UHPLC–HRMS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid 37
and 4 mM ammonium formate in water/acetonitrile as the mobile phase with gradient 38
elution. The matrix effect, recovery, and repeatability, within laboratory 39
reproducibility, and the LODs and LOQs of the r-dSPE cleanup method were 40
investigated. The method showed a good linearity (R2> 0.999) in the ranges of 0.01-1 41
μg/L and 1-100 μg/L for the analyte. The LODs of RB for chili powder and chili oil 42
samples were 0.5 μg/kg. The average recoveries of RB from the samples spiked at 43
four different concentrations (2, 20, 500 and 5000 μg/kg) were in a range from 44
76.7-104.9%. Results showed that the proposed method was simple, fast, economical 45
and effective for the determination of RB in chili powder and chili oil. Considering 46
the excellent sorptive performance of PCX for RB, further work should be done to 47
evaluate the usefulness of the PCX in r-dSPE for the clean-up and analyses of other 48
trace-level alkaline contaminants. 49
Keywords: reversed-dispersive solid phase extraction, chili, rhodamine B, 50
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UHPLC–HRMS/MS 51
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1. Introduction 53
Nowadays, synthetic dyes are sometimes misused and found in foodstuffs since 54
they can preserve or restore the natural color of food products and enhance appeal [1]. 55
Rhodamine B (RB), an important water–soluble xanthene organic dye, has a wide 56
variety of technical applications, such as dyeing silk, wool, jute leather and cotton [2]. 57
However, RB is dangerous if swallowed by human beings and animals, and causes 58
irritation to the skin, eyes and respiratory tract. Its potential health risk to human 59
beings and animals, especially when it is consumed in excessive amounts have been 60
experimentally proven [3]. However, due to its low cost and high effectiveness, RB is 61
still illegally used in some parts of the world. In China, RB is a banned food additive. 62
Therefore, sensitive and reliable methods for the determination of RB in food samples 63
are highly demanded. 64
Only few methods are available for the determination of RB. These methods are 65
mainly based on capillary electrophoresis (CE) [4] and liquid chromatography (HPLC) 66
coupled with other detectors such as mass spectrometers (MS) and fluorescence 67
detection (FLD) [5-8]. Recently, a HPLC-FLD method for the determination of RB in 68
chili-containing products has been published [9]. The MS detectors are more 69
frequently used than FLD's because they provide higher characterization ability 70
through the use of mass spectra of compounds in combination with their retention 71
times. In addition, MS detectors provide higher sensitivity and selectivity than other 72
detectors. However, the occurrence of matrix effects in LC-MS/MS is well known and 73
the removal of interference and minimization of matrix effect is the key to an accurate, 74
robust and sensitive quantitative assay [10]. The cleanup is considered to be the most 75
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laborious but effective process to overcome ion suppression. The cleanup methods 76
including solid phase extraction (SPE) [8, 11], gel permeation chromatography (GPC) 77
[12], cloud point extraction [7], etc. are widely used to solve the matrix effects in 78
complex matrices for the residue analysis of RB. Although SPE had been verified to 79
be effective for cleaning the complex matrix with high pre-concentration factors and 80
simplicity of phase separation, it is relatively expensive, time-consuming and tedious. 81
Recent research activities are being focused on the development of efficient, rapid, 82
economical, and miniaturized sample preparation methods. A dispersive solid phase 83
extraction based on a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) 84
pattern for sample cleanup has been widely developed to improve sample throughput 85
for residue analysis [13, 14]. However, the purification effect is obviously worse than 86
that of SPE. Reversed-dispersive solid phase extraction (r-dSPE) can be categorized 87
as a dispersive solid phase extraction (dSPE) or SPE technique. The r-dSPE exhibits 88
some advantages over traditional dSPE (fewer matrix effects) and SPE (such as 89
without complex equipment; short time requirement and less solvent consumption) 90
[13-15]. Graphene and carbon nanotubes (CNTs) are commonly used as materials for 91
application in the sorbent phase of such processes. Based on the mixed-mode cation 92
exchange (MCX) SPE method, the cation exchange polymer material would probably 93
become a powerful adsorbent to carry out r-dSPE method. Polymer cation exchange 94
(PCX), as a high molecular weight polymer, can adsorb alkaline chemical substances 95
directly and provide an effective separation. As shown in Figure S1 (Supplementary 96
data), RB with quaternary ammonium salt structure was easily adsorbed to PCX based 97
on its characteristics of strong anion structure. In this study, PCX was tried to serve as 98
a sorbent for r-dSPE cleanup method in RB residue analysis from chili powder and 99
chili oil. To the best of our knowledge, this paper was the first to introduce application 100
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of PCX as a sorbent in r-dSPE for sample preparations of RB. 101
2. Materials and Methods 102
2.1. Chemicals and reagents 103
Acetonitrile and methanol (HPLC grade) were obtained from Fisher Scientific 104
(USA). Ammonium hydroxide (HPLC grade) was purchased from Tedia (Weston, 105
America). Ultra-pure water was prepared from a Milli-Q Plus system at 18.2 MΏ 106
(Millipore, Bedford, MA, USA). Cleanert® PCX powder was obtained from Agela 107
Technologies (Tianjing, China). Rhodamine B (≥97.0% purity) was purchased from 108
Sigma (St. Louis, MO, USA). 109
2.2. Samples collection and preparation 110
The chili powder and chili oil samples were obtained from the local markets of 111
Beijing in China. The chili powder samples were homogenized before being analyzed. 112
Blank samples of chili powder and chili oil were collected and analyzed without RB. 113
Spiked samples at four concentration levels (2, 20, 500 and 5000 μg/kg) were 114
prepared by adding the standard solutions to blank chili powder and chili oil. In order 115
to achieve uniformity of the RB in spiked samples, the spiked samples were first 116
vortexed for 5 min, and then placed for 6 hours at room temperature before sample 117
preparation. 118
2.2.1. Chili powder 119
A portion (0.1 g) of sample was accurately weighed into a 15 mL centrifuge tube. 120
After addition of 5 mL of acetonitrile/water (1:1 v/v), the sample was homogenized 121
and ultrasonicated for 5 min at room temperature. To separate the supernatant from 122
the solid sample, the sample was centrifuged at 5000 rpm for 3 min. After 123
centrifugation, 1 mL of the supernatant was transferred into 2 mL eppendorf tube with 124
5 mg PCX, which had been preconditioned with 1 mL acetonitrile. The mixture was 125
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vortexed for 30 s, and then poured off into a simple unit device equipped with 1 mL 126
syringe and 0.22 μm nylon syringe filter. The extracting solution was passed through 127
the unit device manually, and then was washed with 1 mL of acetonitrile. Then, the 128
PCX enriched with the analyte was eluted with 2 mL of ammonium 129
hydroxide/methanol (1:99 v/v). The eluent was collected and transferred into a 130
sampler vial for UPLC-MS analysis. Additionally, the detailed r-dSPE sample 131
preparation procedure is provided in Figure S1 (Supplementary data). 132
2.2.2. Chili oil 133
A portion (0.1 g) of sample was accurately weighed into a 15 mL centrifuge tube. 134
The sample was extracted with 5 mL of acetonitrile and ultrasonicated for 5 min at 135
room temperature, and then centrifuged at 5000 rpm for 3 min. The subsequent 136
procedures were identical to those described in Section 2.2.1. 137
2.3. Chromatographic conditions 138
UPLC analysis was performed on a UPLC Ultimate 3000 system (Dionex) with the 139
column oven temperature maintained at 40 ℃, using an Acquity BEH C18 140
(2.1 mm × 100 mm, 1.7 μm particle size) analytical column (Waters, USA). The 141
aqueous solvent (A) consisted of a mixture of 0.1% of formic acid and 4 mM 142
ammonium formate in water, and the organic phase (B) was acetonitrile with 0.1% 143
formic acid. The gradient started at 50% B was raised to 80% B in the next 3 minutes 144
and then linearly ramped to 100% B in the following 1 minutes. This was followed by 145
re-equilibration at 50% B for 2 minutes prior to the next injection. The flow rate was 146
set to 300 μL/min with a resulting overall runtime of 6 min. The injection volume was 147
5 μL. 148
2.4. Mass spectrometry conditions 149
Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) with 150
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a heated electrospray ionization (HESI) was operated in the positive (ESI+) 151
electrospray ionization modes. The system was controlled by Xcalibur 2.2 (Thermo 152
Fisher Scientific). The spray voltage was 3.5 kV for the positive mode. The 153
temperature of ion transfer capillary, sheath gas, auxiliary gas, sweep gas and S-lens 154
RF level were set to 325 ℃, 30, 10, 0 (arbitrary units) and 55 V, respectively. The 155
instrument was calibrated in the positive mode every 3 days using the calibration 156
solutions, including caffeine, MRFA, and a mixture of fluorinated phosphazines 157
ultramark 1621, provided by the instrument manufacturer. 158
The Q-Exactive detector was operated in targeted single ion monitoring 159
(tSIM)/dd-MS2 (Top N) mode. By tSIM /dd-MS2 (Top N) mode, tSIM spectra were 160
acquired at mass resolving power of 70000 full width at half-maximum (FWHM) in 161
an isolation window of 4 Da without use of any locked mass. Data-dependent 162
acquisition of tandem mass spectra was triggered automatically using an inclusion list 163
that comprised information on m/z values and retention times (RT). Fragmentation 164
mass spectra were recorded at a mass resolving power of 17500 FWHM with the use 165
of a normalized collision energy (NCE) of 35% and a quadrupole isolation window of 166
4 Da. Using this scan mode, the parent ions were selected in the quadrupole 167
(443.22949 m/z for RB) for quantitative analysis by tSIM. The qualitative analysis 168
was performed by dd-MS2 (Top N) with all fragmented ions (399.16928, 355.10712 169
m/z for RB) originating from the parent ion. 170
2.5. Method validation 171
Validation of the method was based on the European Commission Decision 172
2002/657/EC in terms of selectivity, linearity, precision, recovery, matrix effects, 173
limit of detection (LOD), and limit of quantification (LOQ) [16]. 174
To verify the absence of interfering substances around the retention time of RB, 10 175
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blank samples for each kind of sample were prepared and analyzed in accordance 176
with Section 2.2. The matrix interferences were checked compared with the elution 177
time of the analyte. 178
Method linearity was evaluated using matrix matched calibration (MMC) curves, 179
by spiking blank samples at seven concentration levels for the two different matrices. 180
Two series of calibration curves were prepared for each matrix with concentrations 181
corresponding to 0.01-1.0 μg/L and 1.0-100.0 μg/L. Calibration curves were obtained 182
by plotting the peak area versus the analyte concentration. The coefficient of 183
determination (R2) was determined by means of the least square approach. The LOD 184
and LOQ were estimated for a signal-to-noise (S/N) ratio of more than 3 and 10 185
respectively from the chromatograms of samples spiked at the lowest concentration 186
validated. Precision (intra-day repeatability and inter-day reproducibility, in terms of 187
% RSDr and RSDR) and accuracy (percentage recoveries) were estimated by recovery 188
experiments in chili powder and chili oil samples. Intra-day repeatability of the 189
method was evaluated by spiking the standard solutions to the six blank matrices at 190
four different concentration levels (2, 20, 500, 5000 μg/kg) and analyzing in the same 191
run of the day on the LC-MS. The four spiked concentration levels (with 0.1 g sample) 192
yielded the analysis concentrations of 0.02, 0.2, 5 and 50 μg/L after the sample 193
preparation as described in Section 2.2. For inter-day reproducibility, the four 194
concentrations were analyzed in three different days. 195
Matrix effect (ME) was determined by constructing calibration curves in blank 196
extract and in the pure solvent. The effects were expressed in terms of signal 197
suppression/enhancement (SSE) and calculated as follows: SSE = slope of spiked 198
extract/slope of pure solvent standard. 199
3. Results and discussion 200
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3. 1. Sample preparation 201
To detect trace levels of RB residue in chili powder and chili oil, it is necessary to 202
remove pigments and fat from matrix samples. The main challenge of developing a 203
cleanup method was the separation of the analyte of interest from the matrix. 204
RB was usually extracted from chili powder using acetonitrile or methanol, and 205
then cleaned up with SPE after a time-consuming concentration step [4, 8]. However, 206
the analyte was difficult to be extracted completely from the solid samples using 207
organic solvent without water [6]. So the extraction efficiency was first investigated 208
by using acetonitrile and acetonitrile/water (1:1 v/v) in a real positive chili powder 209
sample at 7.042 mg/kg obtained from the market in China. The obtained results 210
showed that the extraction efficiency was better with acetonitrile/water (1:1 v/v) (with 211
the recovery of 97%) than with pure organic solvent (with the recovery of 78%). 212
Therefore, a complete extraction was achieved with 5 mL of acetonitrile/water (1:1 213
v/v), which was consistent with the result of reference [6]. Addition of water increases 214
the solubility of analyte and enhances its homogenization and permeability in the 215
solid sample. Secondly, the parameters that affected the extraction efficiency of RB, 216
such as the amount of PCX, the adsorption time and the type of eluent were carefully 217
studied for r-dSPE cleanup procedure. 218
3. 1.1. Effect of amount of PCX 219
The effects of the amount of PCX (5-50 mg) on recovery were carefully 220
investigated at 10 mg/kg fortification concentration in blank chili powder and chili oil 221
using a 30 s adsorption time. The results are shown in Fig. 1a. The results revealed 222
that the recoveries of RB were above 95% when the amounts of PCX were between 5 223
and 50 mg. It means that 5 mg of PCX sorbent was enough to retain RB. 224
3.1.2. Effect of adsorption time 225
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The effects of the adsorption time for RB were investigated for the chili powder 226
and chili oil extracts at the level of 10 mg/kg at different shaking times (15, 30, 60, 90 227
and 120 s). Fig. 1b showed that a 30 s adsorption time was enough to adsorb RB into 228
PCX. In addition, the r-dSPE procedure based on PCX as sorbent was a fast 229
adsorption process with less adsorption time compared with multi-walled carbon 230
nanotubes (MWCNT) as sorbent [17]. Therefore, a 30 s adsorption time was chosen 231
for all subsequent experiments. 232
3.1.3. Effect of type of eluent 233
As an analytical practice for SPE, it is well known that alkaline substances are 234
easily eluted from the strong cation exchange column in alkaline condition. Therefore, 235
ammonium hydroxide in methanol had been used to release RB after the cleanup 236
procedure with PCX based on the anion exchange retention mechanism. As a result, 237
the methanol combination with varying concentrations of ammonium hydroxide was 238
used to release RB from PCX. The results are shown in Fig. 1c. As expected, as the 239
concentration of ammonium hydroxide increased, higher extraction efficiency was 240
found. However, when more than 1% ammonium hydroxide in methanol was used, no 241
additional enhancement was observed. Therefore, 1% ammonium hydroxide in 242
methanol was chosen as the eluent in the present study. 243
Under all the optimised conditions, the pretreatment procedure by the PCX cleanup 244
was simple and fast. Furthermore, the cost of the proposed cleanup method by PCX 245
adsorbent for one sample is low enough (approximately 0.10 US dollars) compared to 246
that of a solid phase extraction column (at least 3.0 US dollars for each). In addition, 247
the total time required for the cleanup of one sample was only approximately 3 min 248
and far less than that of the SPE method (at least 20 min) [6]. 249
3.2. Optimization of MS acquisition modes 250
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Full scan and tSIM modes are two kinds of commonly used quantitative models for 251
Q Exactive. In this experiment, full scan and tSIM modes were evaluated by the 252
signal to noise (S/N) ratios. The signal to noise (S/N) ratios of RB at the LOD (0.5 253
μg/kg for chili powder) in different acquired modes were shown in Figure S2 254
(Supplementary data). This figure highlights the fact that a narrower mass range in 255
tSIM modes using the quadrupole increases the S/N ratio considerably, which leads to 256
better method detection limits. Therefore, the tSIM mode was selected as the 257
quantitative model for RB. 258
3.3. Method validation 259
The specificity was evaluated by analyzing 10 blank samples in chili powder and 260
chili oil. With the high resolving power and accurate mass measurements of high 261
resolution mass spectrometry, no interfering peak at the retention time of the analyte 262
was observed from the chili powder and chili oil samples. Extracted ion 263
chromatographs for RB in the blank chili powder, blank chili oil and matrix-matched 264
standard at 0.02 μg/L were shown in Fig. 2. 265
Two series of standards calibrations showed satisfactory linearity in the studied 266
ranges with correlation coefficients ≥0.999). Fig. 3 showed the standard curves of 267
chili oil matrix-matched calibration standards with concentrations corresponding to 268
0.01-1.0 μg/L and 1.0-100.0 μg/L. This range was equivalent to 0.001-10 mg/kg in the 269
sample. The LOD and LOQ values of the analyte were 0.5 and 1.5 μg/kg in chili 270
powder, 0.5 and 2 μg/kg in chili oil (Table 1). 271
The results of the recovery experiment (Table 2) showed that the overall average 272
recovery at four different concentrations for chili powder were in the range of 76.7 to 273
94.8%, with RSDr from 2.4 to 10.4% and RSDR from 1.8 to 7.8%, and in the range of 274
79.6 to 104.9%, with RSDr from 1.5 to 4.2% and RSDR from 3.3 to 6.7% for chili oil. 275
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In this study, matrix effects were evaluated for r-dSPE cleanup method and MCX 276
SPE method as described in Section 2.5. The slope ratios for chili powder and chili oil 277
samples were 0.93 and 0.84 for r-dSPE cleanup method. However, slope ratios from 278
SPE cleanup method for chili powder and chili oil samples were 0.89 and 0.78. It can 279
be concluded that there was a slight matrix effect for RB in chili powder and chili oil 280
for r-dSPE cleanup method (a signal suppression or enhancement effect was 281
considered tolerable if the value was 0.8–1.2) [18], and r-dSPE method yielded fewer 282
matrix effects than the SPE method under the same enrichment factor (1.0). In order 283
to compensate the matrix effects and quantify accurately for concentrations of RB in 284
different matrixes, matrix-match calibrations curves were adopted for quantification 285
and were calculated by the external calibration curves. 286
3.4. Applications of the method 287
The validated method was applied to the analysis of RB in 12 chili powders and 10 288
chili oils samples from the local markets using the above method. RB was not 289
detected in these samples. 290
4. Conclusions 291
In the present study, a novel reversed dispersive solid phase extraction method 292
using PCX sorbent based on the high resolution mass spectrometry (Q Orbitrap) was 293
established for the rapid analysis of RB in chili powder and chili oil. The advantages 294
of the proposed cleanup method include the rapidness (3 min), economy (0.10 US 295
dollars) and convenience, with high precision, sensitivity and repeatability. Therefore, 296
the newly developed r-dSPE cleanup method based on PCX sorbent material is 297
expected to be widely applied for the analysis of alkaline contaminants at trace levels 298
in the future for sample cleanup, aside from SPE cleanup method. 299
Acknowledgements 300
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This work was financially supported by National Support Program for Science 301
and Technology (2012BAK01B01) and the International Science and Technology 302
Cooperation Program of China (2011DFA-31770). The authors wish to thank Thermo 303
Fisher Scientific for technical support. 304
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366
367
Figure captions 368
Fig. 1 Effects of the r-dSPE cleanup conditions for RB (n = 3). 369
Fig. 2 Extracted ion chromatographs for RB in the blank chili powder, blank chili oil 370
and matrix-matched standard at 0.02 μg/L. 371
Fig. 3 The standard curves of chili oil matrix-matched calibration standards with 372
concentrations corresponding to 0.01-1.0 μg/L and 1.0-100.0 μg/L. 373
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Highlights
PCX was first used as a sorbent in reversed-dispersive solid phase extraction.
The newly developed r-dSPE method is rapid, economical, convenient and
sensitive.
The r-dSPE method was observed to be superior to SPE in economy and matrix
effect.
UHPLC-HRMS was applied in residue analysis for rhodamine B.
*Highlights (for review)
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Figure 1
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Figure 2
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Figure 3
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Table 1 Calibration curve equations, correlation coefficients (R2), LODs, LOQs and SSE for RB
Sample Concentration range
(μg/L) Liner equations R
2
LOD
(μg/kg)
LOQ
(μg/kg)
SSE (%)
r-dSPE SPE
Chili powder 0.01-1 Y = 5.8273E+6X+1.8738E+5 0.9991 0.5 1.5
0.90 0.87
1-100 Y = 5.0095E+6X+1.7511E+6 0.9999 0.96 0.92
Chili oil 0.01-1 Y = 5.4145E+6X+1.0907E+5 0.9992 0.5 2
0.84 0.77
1-100 Y = 4.3845E+6X+3.1908E+6 0.9998 0.84 0.79
Tables
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Table 2 Precision and recovery of RB in chili powder and chili oil
Sample Fortified concentration
(μg/kg) Average recovery (%, n = 6) RSDr (%) RSDR (%)
Chili powder 2 76.7 10.4 7.8
20 87.1 6.9 5.1
500 88.3 2.4 3.4
5000 94.8 2.4 1.8
Chili oil 2 79.6 4.2 6.7
20 93.7 5.4 3.4
500 85.2 3.7 5.4
5000 104.9 1.5 3.3