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Upconversion luminescence nanoparticles-based immunochromatographic assay for quantitative detection of triamcinolone acetonide in cosmetics
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 214 (2019) 302–308
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Upconversion luminescence nanoparticles-based immunochromatographic assay for quantitative detection of triamcinolone acetonide in cosmetics Shiwei Zhang a,⁎, Tianqi Yao a, Shifeng Wang a, Ronghu Feng a, Liqiong Chen b,⁎, Vivian Zhu c, Guiping Hu a, Heng Zhang a, Guowu Yang a a b c
Shenzhen Academy of Metrology and Quality Inspection, National Nutrition Food Testing Center, Shenzhen 518102, PR China Shenzhen Technology University, Shenzhen 518000, PR China Shenzhen Mingde Experimental School, Shenzhen 518000, PR China
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Article history: Received 31 August 2018 Received in revised form 16 January 2019 Accepted 16 February 2019 Available online 19 February 2019 Keywords: Upconversion luminescence nanoparticles Triamcinolone acetonide Immunochromatography Quantitative
a b s t r a c t Triamcinolone acetonide (TCA) abuse in cosmetics is a common phenomenon. A rapid lateral flow immunochromatographic assay (ICA) was developed for the quantitative detection of TCA using a probe based on upconversion luminescence nanoparticles. Lanthanide-doped upconversion nanoparticles (UCNPs) were synthesized in a system comprising water and ethylene glycol, and a silicon dioxide layer was covered at the carboxyl site. A binding site protection strategy was used to decrease the background signal of UCNPs-ICA. Using dexamethasone derivative as a coating antigen, the optimal UCNPs–ICA exhibits good dynamic linear detection for TCA in the range 1.0–100 ng mL−1 with a median inhibitory concentration of 9.8 ng mL−1. The detection limits for TCA in a cosmetic sample are 20 μg kg−1. The pretreatment of samples only needs dilution with water, suggesting the assay can quantitate TCA on-site using a portable upconversion luminescence reader with a cumulative analysis time of only 10 min. © 2019 Published by Elsevier B.V.
1. Introduction Triamcinolone acetonide (TCA, Fig. 1a), 9-fluor-11β,21-dihydroxy16α,17α-isopropylidenedioxypregna-1,4-diene-3,20 dione, is a potent and long-action glucocorticoid used frequently in therapy of rheumatic, allergic, and dermatologic symptoms [1]. Clinical studies have shown significantly enhanced use of glucocorticoids for the treatment of patients with skin problems, such as psoriasis and eczema [2]. Glucocorticoids reduce inflammation and can temporarily relieve the symptoms of inflammatory skin problems of severe plaque psoriasis. However, they cause severe potential side effects such as permanent skin atopy, pustular psoriasis, blood vessel expansion, and formation of red spots. They have also shown some systematic side effects such as hypertension, diabetes mellitus, osteoporosis, allergic contact dermatitis, Cushing's syndrome, and other side effects. Long-term use and high dosage of these substances increase the risk of side effects, especially if used without medical supervision. These steroids should be kept away from the face, eyes, nose, and mouth. In addition, chronic use of potent topical glucocorticoids can be highly risky, especially for babies and children [3]. Due to these aforementioned issues, glucocorticoids ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Zhang), [email protected] (L. Chen).
https://doi.org/10.1016/j.saa.2019.02.053 1386-1425/© 2019 Published by Elsevier B.V.
should not be used in cosmetic products. However, many cosmetic samples, especially online products, contain glucocorticoids. 30% positive samples of glucocorticoids were added TCA illegally. TCA in cosmetic samples has been detected by various analytical techniques such as reversed-phase high-performance liquid chromatography [4], liquid chromatography-quadrupole-time of flight mass spectrometry [5], matrix-assisted laser desorption/ionization mass spectrometry [6], and LC-tandem mass spectrometry [7–9]. However, these methods are not suitable for on-site detection, as the analytical instruments involved are not portable and the process is time-consuming and laborious. Immunochromatographic assay (ICA) provides a simple and fast on-site detection method to detect glucocorticoids such as dexamethasone (DEX) [10] and hydrocortisone [11]. However, existing immunochromatographic assay used colloidal gold as probe. The assay results can be directly viewed by eye, but have some shortcomings such as inaccurate quantitative results and narrow linear range. More importantly, the sensitivity is unsatisfactory, because high sample concentration is required for acceptable low limit of detection (LOD). In recent years, lanthanide-doped upconversion nanoparticles (UCNPs) have become research focuses in bioassay owing to their attractive optical and chemical features [12–15]. They are sensitive reporter able to emit higher energy light under excitation of lower energy
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Fig. 1. The synthetic route of antigen (a) and modified UCNPs (b).
light. The important advantages of UCP reporters compared to other fluorescent labels include lack of auto fluorescence and a light scattering background, photochemical stability, low toxicity and blinking, a large Stokes shift (fluorescing strongly in the visible region under excitation by near-infrared light), and high resistance to photobleaching [16,17]. In this study, we report the development of a one-step UCNPs-ICA sensor for sensitive, rapid, and on-site quantitative determination of TCA with a testing time of b10 min. 2. Materials and methods 2.1. Materials and reagents C 6 H 17 O 10 Yb (99.9% purity), NH 4 F (99.99% purity), polyethyleneimine (PEI), ethylene glycol (EG) (99.8% purity), diethylenetriaminepentaacetic acid (DTPA) (99.9% purity), (3trimethoxysilylpropyl) diethylenetriamine, N-hydroxysuccinimide (NHS) (98% purity), 1-ethyl 3-[3-(dimethylamino) propyl] carbodiimide (EDC) (98% purity), keyhole limpet hemocyanin (KLH) bovine serum albumin (BSA), and poly (acrylic acid) (PAA) were purchased from Sigma-Aldrich (St Louis, MO, USA). All of the chemicals were of analytical grade. A Milli-Q purification system (Millipore) was used for purified water. All other chemical reagents were of analytical grade or better. Nitrocellulose (NC) membranes (CN140) were obtained from Sartorius (Göttingen, Germany). The antibody was purified using a Carboxy Link Coupling Plus Immobilization Kit (Pierce, Thermo Scientific). 2.2. Apparatus A fluorescence reader (Niutaier. China) was modified to function as upconversion luminescence reader by using a 300 mW, 980 nm laser instead of LED light as the excitation light source. Upconversion fluorescence spectra were measured using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) modified with a 2 W 980 nm laser. Sigma 300 transmission electron microscope (TEM) (Carl Zeiss, Germany) was used for characterizing UCNPs. A XYZ3060
platform comprised motion control with Biostrip Dispenser HGS102 and Airjet HGS102 (BioDot, USA). A fluorescence camera invented in our lab with 2 W 980 nm laser as the excitation light source and as 540 ± 15 nm optical filter as the camera lens was used for the studies. 2.3. Antigen and monoclonal antibody preparation TCA derivative (TCA-COOH) and DEX derivative (DEX-COOH) used as hapten were prepared by the introduction of alkyl chain spacers with pendent carboxylic acid, in the hydrogen of 21-position hydroxyl. The procedure for the synthesis of hapten is shown in Fig. 1a. TCA or DEX was dissolved in pyridine (5 mL) in a 25-mL round-bottom flask to make 1 M solution, followed by adding succinic anhydride (3 mM). The mixture was stirred for 6 h at 60 °C, followed by adding 10% hydrochloric acid/ice-water (V/V) to the organic phase. A white solid thus precipitated was filtered, isolated, and washed thrice with water and dried in a vacuum oven at 50 °C for 8 h. The structure of the hapten was confirmed by MS. TCA-COOH and DEX-COOH was conjugated to KLH and BSA to prepare immune antigen and coating antigen, respectively [18]. Monoclonal antibodies were prepared following Wang et al. method [19]. 2.4. Synthesis of UCNPs derived from NaYF4:Yb3+ and Tm3+ NaYF4:19% Yb and 2% Er UCNPs were synthesized by Zhao's one-pot route [20]. 10 mmol NH4F and 10 mmol NaOH were dissolved in 3 mL of water and 4.5 mL of EG to form solution A. 1 mmol of Re(NO3)3 (Re = Y, Yb, and Er in a molar ratio of 79:19:2) and 0.20 g of PEI were dissolved in 3 mL of water and 4.5 mL of EG to form solution B. Both solutions A and B were stirred for 1 h and then mixed to prepare homogenized solution at room temperature. The mixture was stirred for 10 min, transferred to a Teflon-lined autoclave, and the temperature was immediately increased to 200 °C and maintained at this temperature for 10 h, and then cooled down to room temperature. The resulting nanoparticles were precipitated by adding ethanol, washed three times with ethanol
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followed by water, collected by centrifugation, and finally redispersed in water for future use. 2.5. Modification of UCNPs Fig. 1b shows the procedure for the modification of UCNPs. The NaYF4:Yb,Er UCNPs were coated with a 15-nm silica layer by the microemulsion method [21]. The surface of the NaYF 4 : Yb, Er@SiO 2 nanoparticles was initially modified with amino groups [22]. In a typical process, 0.02 mmol of NaYF4:Yb,Er@ SiO2 nanoparticles were dispersed in 10 mL of water containing 200 μL of acetic acid, followed by adding 20 μL of (3-trimethoxysilylpropyl) diethylenetriamine. The resulting solution was stirred for 4 h. The amino-modified nanoparticles thus formed were collected by centrifugation and washed twice with distilled water. NaYF4:Yb,Er@SiO2-NH2 (0.5 g) was dispersed in 100 mL 0.5% DTPA water solution (pH 6.0), followed by adding 0.04 g NHS and 0.15 g EDC and stirring for 4 h. The carboxyl-modified nanoparticles were collected by centrifugation, washed twice with distilled water twice, and finally dispersed in distilled water. 2.6. Preparation of UCNPs labeled anti-TCA Fig. 2 shows the antibody labeling procedure. TCA–agarose-based conjugates were prepared using a Carboxy Link Coupling Plus Immobilization Kit, following the manufacturers' instructions. The ascites solution was diluted to 5 mg mL−1 (protein concentration) using PBS (0.01 M, pH 7.4). Approximately 2 mL ascites solution and 2 mL of TCA–agarose-based conjugate were added to a spin column. The column was incubated for 60 min at room temperature while rocking to allow binding to occur and then centrifuged. The agarose-based conjugate was washed with 2 mL of 0.01 M pH 7.4 PBS and centrifuged thrice (this step was repeated three times). Approximately 2 mL of 2 mg mL−1 EDC/NHS activated UCNPs (prepared following Wang's method [17]) was added to the spin column containing the agarose-based conjugate. The UCNPs were added in excess for complete labeling of IgG molecules. Subsequently, 10 μL of 5 M sodium cyanoborohydride was added to the reaction and incubated for 2 h at room temperature while rocking to allow binding to occur. The reaction mixture was then centrifuged. The resin was washed with 2 mL of 0.01 M pH 7.4 PBS, and the reaction
mixture was centrifuged. This step was repeated thrice, allowing easy removal of unbound UCNPs. The agarose was eluted with 2 mL of cold elution buffer (0.1 M glycine·HCl at pH 3.0), collected into a centrifuge tube containing 100 μL of neutralization buffer (1 M PBS, pH 9.0), and centrifuged. The precipitate was resuspended in PBS (0.015 M, pH 7.4 containing 1% (w/v) PVP K-40, 1% (w/v) PEG 40000, 1% (w/v) trehalose, and 2% (w/v) BSA). 2.7. Preparation of UCNPs-ICA UCNPs-ICA strips were prepared as follows: DEX-BSA and goat-antimouse IgG were dispensed on separate areas of a NC membrane, designated as the test line (T-line) and calibration line (C-line), using an automatic dispenser delivering a volume of 1 μL cm−1. The conjugate pad was pretreated using PBS (0.015 M, pH 7.4) containing 1% (w/v) PVP K40, 1% (w/v) PEG 40000, 1% (w/v) trehalose, 2% (w/v) BSA. The anti-TCA monoclonal antibody conjugated UCNPs were dispensed into various volumes onto the conjugate pad using an automatic dispenser set. A PVC plate served as the plastic support for the test strip. The NC membrane, conjugate pad, sample pad, and absorbent pad were laminated and pasted onto the PVC plate before it was cut into 2.85-mm-wide and 60-mm-long strips. 2.8. The performance of the UCNPs-ICA Various concentrations of TCA solution were prepared, and a total of 80 μL of solution was applied to the sample pad. After 5 min, the fluorescence intensity of UCNPs-ICA was measured using the upconversion luminescence reader. The integral area of the T-line (FIT) or C-line (FIC) within a fixed peak width was used to express the fluorescence intensity of each line. Standard curve was established using FIT/FIC value and TCA concentration as the abscissa and ordinate, respectively. 2.9. Detection of TCA in spiked samples Cosmetic samples, such as cream, mask and essence, were used in a spiking study. A 0.1 g sample was diluted 20 times with distilled water. 80 μL of diluted sample was used in UCNPs-ICA.
Fig. 2. Binding site protection procedure for conjugating antibody to UCNPs.
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Fig. 3. TEM image and fluorescence scanning graph, showing the preparation of UCNPs. a) TEM image of 55 nm UCNPs, b) TEM image of 150 nm UCNPs, c) silicon dioxide covered and carboxyl site modified 150 nm UCNPs, d) comparison of the fluorescence intensity for the three UCNPs.
3. Results and discussion
3.2. Protecting binding site label
3.1. Characterization of UCNPs
UCNPs are very sensitive fluorescence probes, and they may generate a high background signal, easily interfering with the assay sensitivity and stability. Avoiding unnecessary UCNPs into the assay system would decrease the background signal. Thus, purified antibody and optimizing antibody/UCNPs ratio are required in UCNPs-ICA strip preparation. Proteins A and G can remove most of the impurities, but abundant IgG will
The hexagonal phase NaYF4 has been reported as one of the most efficient hosts for performing infrared to visible photon conversion when activated by Yb3+ and Er3+ ion pairs [23]. One-pot route was developed to synthetize the NaYF4:Yb3+ and Er3+ UCNPs. Ultrapure water and ethylene glycol were chosen as the reaction solvent instead of oleic acid, oleylamine, and octadecene. The particle size of NaYF4:Yb3+ and Tm3 + UCNPs were well controlled by adding different amounts of ultrapure water into absolute ethylene glycol solvent [24] by adjusting the solvent ratio of ultrapure water to ethylene glycol as 2/1 and 1/1.5 (v/v) to obtain 55 nm and 150 nm wavelength, respectively. The TEM images are shown in Fig. 3a and b. The fluorescence intensity of 150 nm UCNPs was approximately 2.5 times than that of 55 nm UCNPs (Fig. 3d). Thus, 150-nm UCNPs were selected to modify silicon dioxide layer. Most of UCNPs luminescent probes used in ICA were synthesized directly by the ligand exchange method [12–15]. Because of complex matrix of cosmetic samples, a coating of silicon dioxide layer is beneficial in preventing matrix interference to fluorescence. More importantly, UCNPs in ICA strip will be agglomerated easily after a certain period of time. The coating a silicon dioxide layer and grafting multi-carboxyl sites can increase the dispersibility of UCNPs in water to prolong the shelf life of ICA strip. The size and morphology of the pristine and surface modified NaYF4:Yb,Er@SiO2-COOH UCNPs were characterized by TEM (Fig. 3c). The UCNPs are well dispersed and uniform in size with an average diameter of approximately 165 nm, which is slightly bigger than the unmodified one. The fluorescence intensity of the modified UCNPs did not decrease significantly (Fig. 3d).
Fig. 4. UCNPs-ICA strip scanning result using the optimized labeled antibody and direct labeled antibody.
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procedures hardly control the labeling site in an antibody. This study provides an easily controlled method to conjugate UCNPs with antibody. Immobilized antigen was used to catch specific IgG and protect the binding site. Abundant UCNPs avoided several antibodies conjugate one UCNPs. Uncombined antibodies and UCNPS were easily separated. Thus, the method ensured only specific and active antibody to conjugate UCNPs, avoided the inactivation of the antibody in conjugation process and increased the average fluorescent intensity of single antibody. Fig. 4 shows the comparison between the binding site protection procedure and traditional method (labeling ascites directly). Background interference and sensitivity of our method are superior to those of the traditional methods. The inhibition ratios increased from 59% (ascites labeled directly) to 86% (optimized label). The strategy is reproducible, because of easy availability of required reagents and kits. It can be used not only for the preparation of UCNPs-antibody conjugation, but also in other biolabels. 3.3. Optimization of reaction time
Fig. 5. Optimization of the test time for the UCNPs-ICA to detect TCA.
exist. Optimizing antibody/UCNPs ration is another tedious process, which needs to be optimized. One UCNP may conjugate to several antibodies with high antibody/UCNPs dosing ratio condition. Conversely, some UCNPs would not conjugate to antibody. Both of the conditions would damage the sensitivity of the assay. Moreover, direct-labeling
UCNPs-ICA reaction time was an important parameter, affecting the fluorescent response. Fig. 5 shows the responses of the UCNPs-ICA to 9.0 ng mL−1 TCA for different periods of time. Responses to the native sample were determined in parallel to use as a control for the experiment. The fluorescence intensity of UCNPs-ICA was collected using the upconversion luminescence reader. To measure the fluorescence level of the T-line, the ratio of the fluorescence intensities of the T-line vs. C-line (FIT/FIC) was determined. The fluorescent signal intensity initially decreased sharply and then became constant. Taking into account the
Fig. 6. a) The portable upconversion luminescence reader. b) The UCNPs-ICA at various TCA concentrations captured using a fluorescence camera. c) Peaks of UCNPs-ICA at various TCA concentrations collected using an upconversion luminescence reader. d) The standard curve determined for the UCNPs-ICA, with varying concentrations of TCA. Each value is the mean of three replicates.
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Table 1 Cross-reactivity of antibody with various chemicals. Competitor
IC50 (ng mL−1)
Cross-reactivity (%)
Competitor
IC50 (ng mL−1)
Cross-reactivity (%)
TCA Cortisone Prednisolone Beclomethasone Betamethasone Meprednisone Prednisolone
9.7 N1000 N1000 N1000 N1000 N1000 N1000
100% b1% b1% b1% b1% b1% b1%
Hydrocortisone Prednisone Dexamethasone Triamcinolone Fludrocortisone Prednisone Beclomethasone
N1000 N1000 N1000 N1000 N1000 N1000 N1000
b1% b1% b1% b1% b1% b1% b1%
time consumed and signal intensity stability, 5 min was routinely used for future experiments. 3.4. Selection of coating antigen Glucocorticoids are a big drug family with similar structure and can be derived from the carboxyl site using a simple synthetic method. Thus, studying the structure–function relationship between antigen and antibody will be convenient. Usually, heterogeneous coating can provide higher sensitivity in competitive immunoassay [25]. In this study, four carboxyl derivatives from TCA, DEX, hydrocortisone prednisone, and betamethasone were prepared and conjugated to BSA for coating antigen material. The antibody recognized the four antigens, but the sensitivity of UCNPs-ICA was significantly different in all the cases. The IC50 values of UCNPs-ICA derived from TCA, DEX, hydrocortisone prednisone, and betamethasone were 87 ng mL−1, 9.8 ng mL−1, 23 ng mL−1 and 46 ng mL−1, respectively, suggesting DEX-COOH-BSA to be an optimal coating antigen. 3.5. Analytical performance of UCP-ICA sensor UCNPs-ICA responses (Fig. 6b) were captured using a fluorescence camera and a fluorescence reader (Fig. 6a) after allowing the test to run for 5 min. A series of concentrations of TCA in PBS were assayed by the test strips. For increase in the TCA concentration, the fluorescence signals of the T-line gradually decreased, while the signals from the Cline remained constant. FIT equaled to FIC at the TCA concentration as low as 1 ng mL−1 and was determined as the sensitivity of this UCNPs-ICA. For a quantitative result, the fluorescence intensity of UCNPs-ICA was collected using a fluorescence reader (Fig. 6c). More accurately, the ratios of the calculated FIT to FIC (FIT/FIC) were used to determine the fluorescence signals of the T-line. Each reported result is the mean of three parallel runs. Overall, a standard curve of various TCA concentrations was gathered for the UCNPs-ICA, as shown in Fig. 6d. The linear detection range of the UCNPs-ICA was determined as 1–100 ng mL−1, suggesting that UCNPs-ICA is able to detect TCA in an accurate and quantitative manner. The specificity of the antibody was estimated using various frequently used glucocorticoids as competitors (Table 1). The crossreactivity rates were all b0.1% by using UCNPs-ICA as the detection method, although the antibody could conjugate the BSA of the carboxyl derivative of the some glucocorticoids, as mentioned in the above section. Table 2 Analytical results and recoveries of cosmetic samples (n=6). Sample
Fortification level (μg kg−1)
Mean ± S.D. (μg g−1)
Recovery (%)
CV (%)
Cream
160 40.0 160 40.0 160 40.0
183 ± 19 48.0 ± 10.2 179 ± 18 46.6 ± 12.1 182 ± 30 49.2 ± 5.3
114 120 112 116 114 123 117
10.4 20.8 10.1 26.0 16.5 10.8 15.7
Mask Essence Average
3.6. Sample determination TCA is often illegally added to cosmetics. Therefore, three types of cosmetics, cream, mask and essence, were used as a real system to analyze the residues of the drug. Cosmetics contain a large amount of lipid, protein, polysaccharide, emulgator, and other compounds, thus requiring complicated traditional pretreatment such as solid-phase extraction and concentration [26]. The UCNPs-ICA was found to be very sensitive; therefore, only 20 times dilution was only needed to eliminate the matrix interference. The LOD of 20 μg kg−1 met the demand of monitoring TCA in cosmetic [7]. The analysis results are summarized in Table 2. All the coefficients of variation were b15.7%, and the average recovery was 117%, exhibiting a good precision. The upconversion luminescence reader is very small (30 × 30 × 15 cm3) and light (2 kg), fulfilling the requirements of successful on-site quantitative analysis method. 4. Conclusion In this study, a fluorescent reader-based strip test was developed for quantitative and sensitive detection of triamcinolone acetonide in cosmetics. Silicon dioxide covered highly luminescent upconversion nanoparticles are explored as probes in the competitive strip assay. An easy and controllable binding site protection strategy was used to prepare antibody-UCNPs conjugation, which could enhance the sensitivity of the UCNPs-ICA. This assay provides an easy, fast (10 min including pretreatment), and low-cost method that can be applied to the on-site determination of TCA. Acknowledgments This work was supported by the Shenzhen Science and Technology Bureau basic research project (JCYJ20170817162119873) and the State Administration for Quality Supervision and Inspection and Quarantine research project of China (2017QK095). References [1] H.Y. Yu, H.M. Liao, Triamcinolone permeation from different liposome formulations through rat skin in vitro, Int. J. Pharm. 127 (1) (1996) 1–7. [2] S.R. Feldman, B.A. Yentzer, Topical clobetasol propionate in the treatment of psoriasis: a review of newer formulations, Am. J. Clin. Dermatol. 10 (2009) 397–406. [3] J. Fiori, V. Andrisano, LC–MS method for the simultaneous determination of six glucocorticoids in pharmaceutical formulations and counterfeit cosmetic products, J. Pharm. Biomed. Anal. 91 (2014) 185–192. [4] L. Matysova, R. Hájková, J. Šícha, et al., Determination of methylparaben, propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC, Anal. Bioanal. Chem. 376 (2003) 440–443. [5] P. Jin, X. Liang, X. Wu, et al., Screening and quantification of 18 glucocorticoid adulterants from herbal pharmaceuticals and health foods by HPLC and confirmed by LC-Q-TOF-MS/MS, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 35 (2018) 10–19. [6] F.P. Barré, B. Flinders, J.P. Garcia, et al., Derivatization strategies for the detection of triamcinolone acetonide in cartilage by using matrix-assisted laser desorption/ionization mass spectrometry imaging, Anal. Chem. 88 (2016) 12051–12059. [7] Y.S. Nam, I.K. Kwon, Y. Lee, et al., Quantitative monitoring of corticosteroids in cosmetic products manufactured in Korea using LC–MS/MS, Forensic Sci. Int. 220 (2012) e23–e28. [8] E.M. Malone, C.T. Elliott, D.G. Kennedy, et al., Screening and quantitative confirmatory method for the analysis of glucocorticoids in bovine milk using liquid chromatography-tandem mass spectrometry, J. AOAC Int. 93 (2010) 1656–1665.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Upconversion luminescence nanoparticles-based immunochromatographic assay for quantitative detection of triamcinolone acetonide in cosmetics Shiwei Zhang a,⁎, Tianqi Yao a, Shifeng Wang a, Ronghu Feng a, Liqiong Chen b,⁎, Vivian Zhu c, Guiping Hu a, Heng Zhang a, Guowu Yang a a b c
Shenzhen Academy of Metrology and Quality Inspection, National Nutrition Food Testing Center, Shenzhen 518102, PR China Shenzhen Technology University, Shenzhen 518000, PR China Shenzhen Mingde Experimental School, Shenzhen 518000, PR China
a r t i c l e
i n f o
Article history: Received 31 August 2018 Received in revised form 16 January 2019 Accepted 16 February 2019 Available online 19 February 2019 Keywords: Upconversion luminescence nanoparticles Triamcinolone acetonide Immunochromatography Quantitative
a b s t r a c t Triamcinolone acetonide (TCA) abuse in cosmetics is a common phenomenon. A rapid lateral flow immunochromatographic assay (ICA) was developed for the quantitative detection of TCA using a probe based on upconversion luminescence nanoparticles. Lanthanide-doped upconversion nanoparticles (UCNPs) were synthesized in a system comprising water and ethylene glycol, and a silicon dioxide layer was covered at the carboxyl site. A binding site protection strategy was used to decrease the background signal of UCNPs-ICA. Using dexamethasone derivative as a coating antigen, the optimal UCNPs–ICA exhibits good dynamic linear detection for TCA in the range 1.0–100 ng mL−1 with a median inhibitory concentration of 9.8 ng mL−1. The detection limits for TCA in a cosmetic sample are 20 μg kg−1. The pretreatment of samples only needs dilution with water, suggesting the assay can quantitate TCA on-site using a portable upconversion luminescence reader with a cumulative analysis time of only 10 min. © 2019 Published by Elsevier B.V.
1. Introduction Triamcinolone acetonide (TCA, Fig. 1a), 9-fluor-11β,21-dihydroxy16α,17α-isopropylidenedioxypregna-1,4-diene-3,20 dione, is a potent and long-action glucocorticoid used frequently in therapy of rheumatic, allergic, and dermatologic symptoms [1]. Clinical studies have shown significantly enhanced use of glucocorticoids for the treatment of patients with skin problems, such as psoriasis and eczema [2]. Glucocorticoids reduce inflammation and can temporarily relieve the symptoms of inflammatory skin problems of severe plaque psoriasis. However, they cause severe potential side effects such as permanent skin atopy, pustular psoriasis, blood vessel expansion, and formation of red spots. They have also shown some systematic side effects such as hypertension, diabetes mellitus, osteoporosis, allergic contact dermatitis, Cushing's syndrome, and other side effects. Long-term use and high dosage of these substances increase the risk of side effects, especially if used without medical supervision. These steroids should be kept away from the face, eyes, nose, and mouth. In addition, chronic use of potent topical glucocorticoids can be highly risky, especially for babies and children [3]. Due to these aforementioned issues, glucocorticoids ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Zhang), [email protected] (L. Chen).
https://doi.org/10.1016/j.saa.2019.02.053 1386-1425/© 2019 Published by Elsevier B.V.
should not be used in cosmetic products. However, many cosmetic samples, especially online products, contain glucocorticoids. 30% positive samples of glucocorticoids were added TCA illegally. TCA in cosmetic samples has been detected by various analytical techniques such as reversed-phase high-performance liquid chromatography [4], liquid chromatography-quadrupole-time of flight mass spectrometry [5], matrix-assisted laser desorption/ionization mass spectrometry [6], and LC-tandem mass spectrometry [7–9]. However, these methods are not suitable for on-site detection, as the analytical instruments involved are not portable and the process is time-consuming and laborious. Immunochromatographic assay (ICA) provides a simple and fast on-site detection method to detect glucocorticoids such as dexamethasone (DEX) [10] and hydrocortisone [11]. However, existing immunochromatographic assay used colloidal gold as probe. The assay results can be directly viewed by eye, but have some shortcomings such as inaccurate quantitative results and narrow linear range. More importantly, the sensitivity is unsatisfactory, because high sample concentration is required for acceptable low limit of detection (LOD). In recent years, lanthanide-doped upconversion nanoparticles (UCNPs) have become research focuses in bioassay owing to their attractive optical and chemical features [12–15]. They are sensitive reporter able to emit higher energy light under excitation of lower energy
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Fig. 1. The synthetic route of antigen (a) and modified UCNPs (b).
light. The important advantages of UCP reporters compared to other fluorescent labels include lack of auto fluorescence and a light scattering background, photochemical stability, low toxicity and blinking, a large Stokes shift (fluorescing strongly in the visible region under excitation by near-infrared light), and high resistance to photobleaching [16,17]. In this study, we report the development of a one-step UCNPs-ICA sensor for sensitive, rapid, and on-site quantitative determination of TCA with a testing time of b10 min. 2. Materials and methods 2.1. Materials and reagents C 6 H 17 O 10 Yb (99.9% purity), NH 4 F (99.99% purity), polyethyleneimine (PEI), ethylene glycol (EG) (99.8% purity), diethylenetriaminepentaacetic acid (DTPA) (99.9% purity), (3trimethoxysilylpropyl) diethylenetriamine, N-hydroxysuccinimide (NHS) (98% purity), 1-ethyl 3-[3-(dimethylamino) propyl] carbodiimide (EDC) (98% purity), keyhole limpet hemocyanin (KLH) bovine serum albumin (BSA), and poly (acrylic acid) (PAA) were purchased from Sigma-Aldrich (St Louis, MO, USA). All of the chemicals were of analytical grade. A Milli-Q purification system (Millipore) was used for purified water. All other chemical reagents were of analytical grade or better. Nitrocellulose (NC) membranes (CN140) were obtained from Sartorius (Göttingen, Germany). The antibody was purified using a Carboxy Link Coupling Plus Immobilization Kit (Pierce, Thermo Scientific). 2.2. Apparatus A fluorescence reader (Niutaier. China) was modified to function as upconversion luminescence reader by using a 300 mW, 980 nm laser instead of LED light as the excitation light source. Upconversion fluorescence spectra were measured using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) modified with a 2 W 980 nm laser. Sigma 300 transmission electron microscope (TEM) (Carl Zeiss, Germany) was used for characterizing UCNPs. A XYZ3060
platform comprised motion control with Biostrip Dispenser HGS102 and Airjet HGS102 (BioDot, USA). A fluorescence camera invented in our lab with 2 W 980 nm laser as the excitation light source and as 540 ± 15 nm optical filter as the camera lens was used for the studies. 2.3. Antigen and monoclonal antibody preparation TCA derivative (TCA-COOH) and DEX derivative (DEX-COOH) used as hapten were prepared by the introduction of alkyl chain spacers with pendent carboxylic acid, in the hydrogen of 21-position hydroxyl. The procedure for the synthesis of hapten is shown in Fig. 1a. TCA or DEX was dissolved in pyridine (5 mL) in a 25-mL round-bottom flask to make 1 M solution, followed by adding succinic anhydride (3 mM). The mixture was stirred for 6 h at 60 °C, followed by adding 10% hydrochloric acid/ice-water (V/V) to the organic phase. A white solid thus precipitated was filtered, isolated, and washed thrice with water and dried in a vacuum oven at 50 °C for 8 h. The structure of the hapten was confirmed by MS. TCA-COOH and DEX-COOH was conjugated to KLH and BSA to prepare immune antigen and coating antigen, respectively [18]. Monoclonal antibodies were prepared following Wang et al. method [19]. 2.4. Synthesis of UCNPs derived from NaYF4:Yb3+ and Tm3+ NaYF4:19% Yb and 2% Er UCNPs were synthesized by Zhao's one-pot route [20]. 10 mmol NH4F and 10 mmol NaOH were dissolved in 3 mL of water and 4.5 mL of EG to form solution A. 1 mmol of Re(NO3)3 (Re = Y, Yb, and Er in a molar ratio of 79:19:2) and 0.20 g of PEI were dissolved in 3 mL of water and 4.5 mL of EG to form solution B. Both solutions A and B were stirred for 1 h and then mixed to prepare homogenized solution at room temperature. The mixture was stirred for 10 min, transferred to a Teflon-lined autoclave, and the temperature was immediately increased to 200 °C and maintained at this temperature for 10 h, and then cooled down to room temperature. The resulting nanoparticles were precipitated by adding ethanol, washed three times with ethanol
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followed by water, collected by centrifugation, and finally redispersed in water for future use. 2.5. Modification of UCNPs Fig. 1b shows the procedure for the modification of UCNPs. The NaYF4:Yb,Er UCNPs were coated with a 15-nm silica layer by the microemulsion method [21]. The surface of the NaYF 4 : Yb, Er@SiO 2 nanoparticles was initially modified with amino groups [22]. In a typical process, 0.02 mmol of NaYF4:Yb,Er@ SiO2 nanoparticles were dispersed in 10 mL of water containing 200 μL of acetic acid, followed by adding 20 μL of (3-trimethoxysilylpropyl) diethylenetriamine. The resulting solution was stirred for 4 h. The amino-modified nanoparticles thus formed were collected by centrifugation and washed twice with distilled water. NaYF4:Yb,Er@SiO2-NH2 (0.5 g) was dispersed in 100 mL 0.5% DTPA water solution (pH 6.0), followed by adding 0.04 g NHS and 0.15 g EDC and stirring for 4 h. The carboxyl-modified nanoparticles were collected by centrifugation, washed twice with distilled water twice, and finally dispersed in distilled water. 2.6. Preparation of UCNPs labeled anti-TCA Fig. 2 shows the antibody labeling procedure. TCA–agarose-based conjugates were prepared using a Carboxy Link Coupling Plus Immobilization Kit, following the manufacturers' instructions. The ascites solution was diluted to 5 mg mL−1 (protein concentration) using PBS (0.01 M, pH 7.4). Approximately 2 mL ascites solution and 2 mL of TCA–agarose-based conjugate were added to a spin column. The column was incubated for 60 min at room temperature while rocking to allow binding to occur and then centrifuged. The agarose-based conjugate was washed with 2 mL of 0.01 M pH 7.4 PBS and centrifuged thrice (this step was repeated three times). Approximately 2 mL of 2 mg mL−1 EDC/NHS activated UCNPs (prepared following Wang's method [17]) was added to the spin column containing the agarose-based conjugate. The UCNPs were added in excess for complete labeling of IgG molecules. Subsequently, 10 μL of 5 M sodium cyanoborohydride was added to the reaction and incubated for 2 h at room temperature while rocking to allow binding to occur. The reaction mixture was then centrifuged. The resin was washed with 2 mL of 0.01 M pH 7.4 PBS, and the reaction
mixture was centrifuged. This step was repeated thrice, allowing easy removal of unbound UCNPs. The agarose was eluted with 2 mL of cold elution buffer (0.1 M glycine·HCl at pH 3.0), collected into a centrifuge tube containing 100 μL of neutralization buffer (1 M PBS, pH 9.0), and centrifuged. The precipitate was resuspended in PBS (0.015 M, pH 7.4 containing 1% (w/v) PVP K-40, 1% (w/v) PEG 40000, 1% (w/v) trehalose, and 2% (w/v) BSA). 2.7. Preparation of UCNPs-ICA UCNPs-ICA strips were prepared as follows: DEX-BSA and goat-antimouse IgG were dispensed on separate areas of a NC membrane, designated as the test line (T-line) and calibration line (C-line), using an automatic dispenser delivering a volume of 1 μL cm−1. The conjugate pad was pretreated using PBS (0.015 M, pH 7.4) containing 1% (w/v) PVP K40, 1% (w/v) PEG 40000, 1% (w/v) trehalose, 2% (w/v) BSA. The anti-TCA monoclonal antibody conjugated UCNPs were dispensed into various volumes onto the conjugate pad using an automatic dispenser set. A PVC plate served as the plastic support for the test strip. The NC membrane, conjugate pad, sample pad, and absorbent pad were laminated and pasted onto the PVC plate before it was cut into 2.85-mm-wide and 60-mm-long strips. 2.8. The performance of the UCNPs-ICA Various concentrations of TCA solution were prepared, and a total of 80 μL of solution was applied to the sample pad. After 5 min, the fluorescence intensity of UCNPs-ICA was measured using the upconversion luminescence reader. The integral area of the T-line (FIT) or C-line (FIC) within a fixed peak width was used to express the fluorescence intensity of each line. Standard curve was established using FIT/FIC value and TCA concentration as the abscissa and ordinate, respectively. 2.9. Detection of TCA in spiked samples Cosmetic samples, such as cream, mask and essence, were used in a spiking study. A 0.1 g sample was diluted 20 times with distilled water. 80 μL of diluted sample was used in UCNPs-ICA.
Fig. 2. Binding site protection procedure for conjugating antibody to UCNPs.
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Fig. 3. TEM image and fluorescence scanning graph, showing the preparation of UCNPs. a) TEM image of 55 nm UCNPs, b) TEM image of 150 nm UCNPs, c) silicon dioxide covered and carboxyl site modified 150 nm UCNPs, d) comparison of the fluorescence intensity for the three UCNPs.
3. Results and discussion
3.2. Protecting binding site label
3.1. Characterization of UCNPs
UCNPs are very sensitive fluorescence probes, and they may generate a high background signal, easily interfering with the assay sensitivity and stability. Avoiding unnecessary UCNPs into the assay system would decrease the background signal. Thus, purified antibody and optimizing antibody/UCNPs ratio are required in UCNPs-ICA strip preparation. Proteins A and G can remove most of the impurities, but abundant IgG will
The hexagonal phase NaYF4 has been reported as one of the most efficient hosts for performing infrared to visible photon conversion when activated by Yb3+ and Er3+ ion pairs [23]. One-pot route was developed to synthetize the NaYF4:Yb3+ and Er3+ UCNPs. Ultrapure water and ethylene glycol were chosen as the reaction solvent instead of oleic acid, oleylamine, and octadecene. The particle size of NaYF4:Yb3+ and Tm3 + UCNPs were well controlled by adding different amounts of ultrapure water into absolute ethylene glycol solvent [24] by adjusting the solvent ratio of ultrapure water to ethylene glycol as 2/1 and 1/1.5 (v/v) to obtain 55 nm and 150 nm wavelength, respectively. The TEM images are shown in Fig. 3a and b. The fluorescence intensity of 150 nm UCNPs was approximately 2.5 times than that of 55 nm UCNPs (Fig. 3d). Thus, 150-nm UCNPs were selected to modify silicon dioxide layer. Most of UCNPs luminescent probes used in ICA were synthesized directly by the ligand exchange method [12–15]. Because of complex matrix of cosmetic samples, a coating of silicon dioxide layer is beneficial in preventing matrix interference to fluorescence. More importantly, UCNPs in ICA strip will be agglomerated easily after a certain period of time. The coating a silicon dioxide layer and grafting multi-carboxyl sites can increase the dispersibility of UCNPs in water to prolong the shelf life of ICA strip. The size and morphology of the pristine and surface modified NaYF4:Yb,Er@SiO2-COOH UCNPs were characterized by TEM (Fig. 3c). The UCNPs are well dispersed and uniform in size with an average diameter of approximately 165 nm, which is slightly bigger than the unmodified one. The fluorescence intensity of the modified UCNPs did not decrease significantly (Fig. 3d).
Fig. 4. UCNPs-ICA strip scanning result using the optimized labeled antibody and direct labeled antibody.
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procedures hardly control the labeling site in an antibody. This study provides an easily controlled method to conjugate UCNPs with antibody. Immobilized antigen was used to catch specific IgG and protect the binding site. Abundant UCNPs avoided several antibodies conjugate one UCNPs. Uncombined antibodies and UCNPS were easily separated. Thus, the method ensured only specific and active antibody to conjugate UCNPs, avoided the inactivation of the antibody in conjugation process and increased the average fluorescent intensity of single antibody. Fig. 4 shows the comparison between the binding site protection procedure and traditional method (labeling ascites directly). Background interference and sensitivity of our method are superior to those of the traditional methods. The inhibition ratios increased from 59% (ascites labeled directly) to 86% (optimized label). The strategy is reproducible, because of easy availability of required reagents and kits. It can be used not only for the preparation of UCNPs-antibody conjugation, but also in other biolabels. 3.3. Optimization of reaction time
Fig. 5. Optimization of the test time for the UCNPs-ICA to detect TCA.
exist. Optimizing antibody/UCNPs ration is another tedious process, which needs to be optimized. One UCNP may conjugate to several antibodies with high antibody/UCNPs dosing ratio condition. Conversely, some UCNPs would not conjugate to antibody. Both of the conditions would damage the sensitivity of the assay. Moreover, direct-labeling
UCNPs-ICA reaction time was an important parameter, affecting the fluorescent response. Fig. 5 shows the responses of the UCNPs-ICA to 9.0 ng mL−1 TCA for different periods of time. Responses to the native sample were determined in parallel to use as a control for the experiment. The fluorescence intensity of UCNPs-ICA was collected using the upconversion luminescence reader. To measure the fluorescence level of the T-line, the ratio of the fluorescence intensities of the T-line vs. C-line (FIT/FIC) was determined. The fluorescent signal intensity initially decreased sharply and then became constant. Taking into account the
Fig. 6. a) The portable upconversion luminescence reader. b) The UCNPs-ICA at various TCA concentrations captured using a fluorescence camera. c) Peaks of UCNPs-ICA at various TCA concentrations collected using an upconversion luminescence reader. d) The standard curve determined for the UCNPs-ICA, with varying concentrations of TCA. Each value is the mean of three replicates.
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Table 1 Cross-reactivity of antibody with various chemicals. Competitor
IC50 (ng mL−1)
Cross-reactivity (%)
Competitor
IC50 (ng mL−1)
Cross-reactivity (%)
TCA Cortisone Prednisolone Beclomethasone Betamethasone Meprednisone Prednisolone
9.7 N1000 N1000 N1000 N1000 N1000 N1000
100% b1% b1% b1% b1% b1% b1%
Hydrocortisone Prednisone Dexamethasone Triamcinolone Fludrocortisone Prednisone Beclomethasone
N1000 N1000 N1000 N1000 N1000 N1000 N1000
b1% b1% b1% b1% b1% b1% b1%
time consumed and signal intensity stability, 5 min was routinely used for future experiments. 3.4. Selection of coating antigen Glucocorticoids are a big drug family with similar structure and can be derived from the carboxyl site using a simple synthetic method. Thus, studying the structure–function relationship between antigen and antibody will be convenient. Usually, heterogeneous coating can provide higher sensitivity in competitive immunoassay [25]. In this study, four carboxyl derivatives from TCA, DEX, hydrocortisone prednisone, and betamethasone were prepared and conjugated to BSA for coating antigen material. The antibody recognized the four antigens, but the sensitivity of UCNPs-ICA was significantly different in all the cases. The IC50 values of UCNPs-ICA derived from TCA, DEX, hydrocortisone prednisone, and betamethasone were 87 ng mL−1, 9.8 ng mL−1, 23 ng mL−1 and 46 ng mL−1, respectively, suggesting DEX-COOH-BSA to be an optimal coating antigen. 3.5. Analytical performance of UCP-ICA sensor UCNPs-ICA responses (Fig. 6b) were captured using a fluorescence camera and a fluorescence reader (Fig. 6a) after allowing the test to run for 5 min. A series of concentrations of TCA in PBS were assayed by the test strips. For increase in the TCA concentration, the fluorescence signals of the T-line gradually decreased, while the signals from the Cline remained constant. FIT equaled to FIC at the TCA concentration as low as 1 ng mL−1 and was determined as the sensitivity of this UCNPs-ICA. For a quantitative result, the fluorescence intensity of UCNPs-ICA was collected using a fluorescence reader (Fig. 6c). More accurately, the ratios of the calculated FIT to FIC (FIT/FIC) were used to determine the fluorescence signals of the T-line. Each reported result is the mean of three parallel runs. Overall, a standard curve of various TCA concentrations was gathered for the UCNPs-ICA, as shown in Fig. 6d. The linear detection range of the UCNPs-ICA was determined as 1–100 ng mL−1, suggesting that UCNPs-ICA is able to detect TCA in an accurate and quantitative manner. The specificity of the antibody was estimated using various frequently used glucocorticoids as competitors (Table 1). The crossreactivity rates were all b0.1% by using UCNPs-ICA as the detection method, although the antibody could conjugate the BSA of the carboxyl derivative of the some glucocorticoids, as mentioned in the above section. Table 2 Analytical results and recoveries of cosmetic samples (n=6). Sample
Fortification level (μg kg−1)
Mean ± S.D. (μg g−1)
Recovery (%)
CV (%)
Cream
160 40.0 160 40.0 160 40.0
183 ± 19 48.0 ± 10.2 179 ± 18 46.6 ± 12.1 182 ± 30 49.2 ± 5.3
114 120 112 116 114 123 117
10.4 20.8 10.1 26.0 16.5 10.8 15.7
Mask Essence Average
3.6. Sample determination TCA is often illegally added to cosmetics. Therefore, three types of cosmetics, cream, mask and essence, were used as a real system to analyze the residues of the drug. Cosmetics contain a large amount of lipid, protein, polysaccharide, emulgator, and other compounds, thus requiring complicated traditional pretreatment such as solid-phase extraction and concentration [26]. The UCNPs-ICA was found to be very sensitive; therefore, only 20 times dilution was only needed to eliminate the matrix interference. The LOD of 20 μg kg−1 met the demand of monitoring TCA in cosmetic [7]. The analysis results are summarized in Table 2. All the coefficients of variation were b15.7%, and the average recovery was 117%, exhibiting a good precision. The upconversion luminescence reader is very small (30 × 30 × 15 cm3) and light (2 kg), fulfilling the requirements of successful on-site quantitative analysis method. 4. Conclusion In this study, a fluorescent reader-based strip test was developed for quantitative and sensitive detection of triamcinolone acetonide in cosmetics. Silicon dioxide covered highly luminescent upconversion nanoparticles are explored as probes in the competitive strip assay. An easy and controllable binding site protection strategy was used to prepare antibody-UCNPs conjugation, which could enhance the sensitivity of the UCNPs-ICA. This assay provides an easy, fast (10 min including pretreatment), and low-cost method that can be applied to the on-site determination of TCA. Acknowledgments This work was supported by the Shenzhen Science and Technology Bureau basic research project (JCYJ20170817162119873) and the State Administration for Quality Supervision and Inspection and Quarantine research project of China (2017QK095). References [1] H.Y. Yu, H.M. Liao, Triamcinolone permeation from different liposome formulations through rat skin in vitro, Int. J. Pharm. 127 (1) (1996) 1–7. [2] S.R. Feldman, B.A. Yentzer, Topical clobetasol propionate in the treatment of psoriasis: a review of newer formulations, Am. J. Clin. Dermatol. 10 (2009) 397–406. [3] J. Fiori, V. Andrisano, LC–MS method for the simultaneous determination of six glucocorticoids in pharmaceutical formulations and counterfeit cosmetic products, J. Pharm. Biomed. Anal. 91 (2014) 185–192. [4] L. Matysova, R. Hájková, J. Šícha, et al., Determination of methylparaben, propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC, Anal. Bioanal. Chem. 376 (2003) 440–443. [5] P. Jin, X. Liang, X. Wu, et al., Screening and quantification of 18 glucocorticoid adulterants from herbal pharmaceuticals and health foods by HPLC and confirmed by LC-Q-TOF-MS/MS, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 35 (2018) 10–19. [6] F.P. Barré, B. Flinders, J.P. Garcia, et al., Derivatization strategies for the detection of triamcinolone acetonide in cartilage by using matrix-assisted laser desorption/ionization mass spectrometry imaging, Anal. Chem. 88 (2016) 12051–12059. [7] Y.S. Nam, I.K. Kwon, Y. Lee, et al., Quantitative monitoring of corticosteroids in cosmetic products manufactured in Korea using LC–MS/MS, Forensic Sci. Int. 220 (2012) e23–e28. [8] E.M. Malone, C.T. Elliott, D.G. Kennedy, et al., Screening and quantitative confirmatory method for the analysis of glucocorticoids in bovine milk using liquid chromatography-tandem mass spectrometry, J. AOAC Int. 93 (2010) 1656–1665.
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