Rapid Detection of Six Glucocorticoids Added Illegally to Dietary Supplements by Combining TLC with Spot-Concentrated Raman Scattering

molecules

Article Rapid Detection of Six Glucocorticoids Added Illegally to Dietary Supplements by Combining TLC with Spot-Concentrated Raman Scattering

Li Li, Xin Liang * ID , Tao Xu, Feng Xu and Wei Dong College of Pharmacy, Qiqihar Medical University, Qiqihar 161006, China; [email protected] (L.L.); [email protected] (T.X.); [email protected] (F.X.); [email protected] (W.D.) * Correspondence: [email protected]; Tel.: +86-0452-2663172

Received: 16 May 2018; Accepted: 19 June 2018; Published: 21 June 2018 Abstract: The objective of this study was to establish a novel method for rapid detection of six glucocorticoids (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) added illegally in dietary supplements simultaneously by combining thin layer chromatography (TLC) with spot-concentrated Raman scattering (SCRS). The doping ingredients were separated by TLC, and viewed and located with UV light (254 nm), enriched by chromatography, then Raman spectra were directly detected by a Raman Imagine microscope with 780 nm laser source. This method had complementary advantages of TLC and Raman spectroscopy, which enhanced the specificity of the test results. The limit of detection (LOD) of the reference substances were 4 µg, 4 µg, 4 µg, 6 µg, 6 µg, and 4 µg, respectively. The method was used to study the six glucocorticoids added illegally in five dietary supplements. Fake drugs had been detected. The study showed that the TLC-SCRS method is simple, rapid, specific, sensitive, and reliable. The method could be used for effective separation and detection of six chemical components used in dietary supplement products, and would have good prospects for on-site qualitative screening of dietary supplement products for adulterants.

Keywords: thin layer chromatography (TLC); spot concentrated Raman scattering (SCRS); glucocorticoids; dietary supplement; illegally adulterants

1. Introduction A dietary supplement is a commercially available product that is consumed as an addition to the usual diet and includes vitamins, minerals, herbs (botanicals), amino acids, and a variety of other products. Marketing claims for some dietary substances include improvements in overall health status, enhancement of cognitive or physical performance, increase in energy, loss of excess weight, attenuation of pain, and other favorable effects [1]. Recently, the demand for dietary supplements is increasing signiﬁcantly due to the belief that the dietary supplement materials are harmless and have fewer side effects than chemical drugs [2]. Many studies have reported on the adulteration of dietary supplements with synthetic drugs [3–6]. The chemical substances that are most likely added in dietary supplements are glucocorticoids. To seize better effects, some producers added chemical drugs, such as prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone illegally [7–10], posing a severe threat to public health. However, abuse of glucocorticoids in dietary supplements results in an unpredictable risk of illness due to side effects, including the possibility of poor control of the adrenal axis, opportunistic infection, diabetes mellitus, osteoporosis [11], Cushing’s syndrome, pneumonia, asthma, and herpeskeratitis [12,13]. Various analytical techniques used for reliable, high-quality analyses of adulterants in dietary supplements have been developed, including high-performance liquid chromatography-mass

Molecules 2018, 23, 1504; doi:10.3390/molecules23071504 www.mdpi.com/journal/molecules Molecules 2018, 23, 1504 2 of 13 spectrometry (HPLC-MS) [14], gas chromatography-mass spectrometry (GC-MS) [15,16], and high-performance liquid chromatography (HPLC) with ultraviolet-visible detection (UV–VIS) [17,18]. However, the disadvantages of all the methods above include being of high cost, time-consuming, and complicated. Thus, in our study, we chose a high-speed, high-selectivity, low-cost screening method, which combined thin layer chromatography (TLC) with spot-concentrated Raman scattering (SCRS) to directly identify trace adulterants. Raman spectroscopy is not a separation technique, so we could not use it to differentiate several components in a mixture. Among many separation methods, TLC is relatively simple and cheap [19–21]. Compared to other TLC methods, including TLC-dynamic surface enhanced Raman spectroscopy (DSERS) [22–25] and TLC-surface enhanced Raman spectroscopy (SERS), the new method in our study needs no reference substance. The combination of TLC and SCRS is suitable for rapid detection of health supervision when portable Raman spectroscopy is used, due to its simplicity, rapidity, and speciﬁcity. The TLC-SCRS (combining thin layer chromatography with spot-concentrated Raman scattering) method established in this article was successfully applied for rapid on-site detection of six glucocorticoids which adulterated dietary supplements. In this article, a great challenge ahead was that the spot of drugs on the plate was too tenuous, and the Raman signal was too weak, thus, we focused on the concentration of the spot on TLC plate, and aimed to develop a new method to deal with the spots of six glucocorticoids. The objective of this study was to screen for the presence of six glucocorticoids added to dietary supplements by TLC-SCRS.

2. Results and Discussion

2.1. Thin-Layer Chromatography Condition The six reference substance solutions and mixture solution were spotted on one TLC plate and then were eluted in a developing chamber with the optimum mobile phase. After the elution, the TLC plate was evaporated naturally, and the separated spots were then visualized and located under ultraviolet illumination at 254 nm. Since the polarity of the six analytes did not differ substantially, dissimilar developing solvents prepared from a variety of solvents were investigated to achieve complete separation of the six analytes. Through the investigation and optimization of several different developing solvents [26] by detecting the spots under 254 nm, when dichloromethane-acetone-methanol = 12:2:0.5, (v/v/v) was used as the optimum mobile phase, the six analytes were mutually separated (Figure1). The Rf value of prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone on the TLC plate were 0.44, 0.75, 0.25, 0.28, 0.71, 0.35, respectively. Due to the similar structure, some of them had similar Rf values, such as prednisolone and hydrocortisone or prednisone acetate and hydrocortisone acetate. Thus, we could not deﬁne the spot only by the Rf value. Combining the Raman spectra of spots to distinguish the spots having similar Rf value, could enhance experimental speciﬁcity. The Rf value was calculated as follows:

distance of the substance zone from the sample origin (mm) R = (1) f solvent front migration distance (mm)

Note: The distance was measured from the application line to the middle of the substance spots. Spots in the mixture solution should have similar Rf values, shape, and color as spots derived from the standard compounds. The deposition amount on TLC plate was calculated as follows:

Deposition amount (µg) = Concentration (µg/µL) × Volume of spotting (µL) (2) Molecules 2018, 23, x 3 of 13 Molecules 2018, 23, 1504 3 of 13 Molecules 2018, 23, x 3 of 13

A B C D E F M Figure 1. Results from TLC analysisA ofB the sixC referenceD E chemicals F M and mixture solution. A, B, C, D, E, FigureF: reference 1. Results substance from TLC of prednisone,analysis of the prednisone six reference acetate, chemicals prednisolone, and mixture hydrocortisone, solution. A, hydrocortisone B, C, D, E, Figure 1. Results from TLC analysis of the six reference chemicals and mixture solution. A, B, C, D, E, F: referenceacetate, and substance dexamethasone; of prednisone, and prednisoneM: mixture acetate,solution. prednisolone, hydrocortisone, hydrocortisone F: reference substance of prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone; and M: mixture solution. 2.2. acetate,The TLC-SCRS and dexamethasone; Method and M: mixture solution. 2.2. The TLC-SCRS Method 2.2. TheInTLC-SCRS this method, Method a great challenge ahead was the drug spot on the plate usually could not meet theIn demand this method, of Raman a great spectroscopy challenge aheaddetection was sensitiv the drugity; spot we focusedon the plate on the usually drug spot could concentration not meet In this method, a great challenge ahead was the drug spot on the plate usually could not meet the theon demand the TLC of Ramanplate, and spectroscopy aimed to detectiondevelop ansensitiv effectity;ive wemethod focused to onobtain the drug Raman spot signals concentration of the six demand of Raman spectroscopy detection sensitivity; we focused on the drug spot concentration on the on glucocorticoids.the TLC plate, and aimed to develop an effective method to obtain Raman signals of the six TLC plate, and aimed to develop an effective method to obtain Raman signals of the six glucocorticoids. glucocorticoids.Sample solutions (the diameters were no more than 4 mm) were spotted on one TLC plate and Sample solutions (the diameters were no more than 4 mm) were spotted on one TLC plate and elutedSample in a solutions developing (the chamber diameters with were dichloromethan no more thane-acetone-methanol 4 mm) were spotted (12:2:0.5). on one AfterTLC plate the elution, and elutedelutedthe TLC in in a a developingplate developing was evaporated chamber chamber with naturally, with dichloromethan dichloromethane-acetone-methanol the separae-acetone-methanolted spots were then (12:2:0.5).visu (12:2:0.5).alized After and After locatedthe the elution, elution, under thetheultraviolet TLC TLC plate plate illuminationwas was evaporated evaporated at 254 naturally, naturally, nm, and the theafter separa separated thatted we spots spotsconcentrated were were then then the visu visualized drugalized spot and and on located locatedthe TLC under under plate, ultravioletultravioletfollowing illumination illuminationthe steps with at at 254ethanol 254 nm, nm, (shownand and after after in thatFigure that we we 2I). concentrated concentrated Finally, the theconcentrated the drug drug spot spot spot on on the was the TLC obtained TLC plate, plate, on followingfollowingwhich we the the collected steps steps with with Raman ethanol ethanol signals. (shown (shown in in Figure Figure 2I).2I). Finally, Finally, the the concentrated concentrated spot spot was was obtained obtained on on whichwhich weThe we collected collecteddetailed Raman process Raman signals. signals.of spot concentration on TLC plate was as follows: when we look down at theThe Theplate detailed detailed after location process process under of of spot spot 254 concentration concentrationnm ultraviolet on onillumi TLC TLCnation, plate plate was we was couldas as follows: follows: see the when whenspot onwe we the look look plate down down (step at at 1); thethethen plate plate we after after cut location the location bottom under under edge 254 254 nmof nmthe ultraviolet ultravioletspot with illumi a illumination, knifenation, in awe line wecould that could see is tangent seethe thespot spot toon the the on spot,plate the plate and(step (stepdug 1); a then1);small then we holecut we the cuton bottom the bottomopposite edge edge edgeof the of of spot the the spot withspot with ato knife the a knife depth in a in line that a line that aluminum that is tangent is tangent plate to couldthe to thespot, be spot, seenand and dug(step dug a 2); smallathen small hole 10 hole μonL ethanol onthe theopposite oppositewas spotted edge edge of on the of the thespot point spot to theof to tangency thedepth depth that wi thataluminumth a aluminumcapillary plate (step plate could 3); could the be drugseen be seen (stepin the (step 2); spot µ then2);wasthen 10 eluted μL 10 ethanol Lfrom ethanol wasthe bottomspotted was spotted edge on the to on pointthe the top point of edgetangency of (s tangencytep wi 4);th Raman a with capillary a signals capillary (step were 3); (step thecollected 3); drug the in drugon the the inspot drug- the wasspotconcentrated eluted was elutedfrom parts the from bottom (step the bottom5); edge finally, to edge the the top to concentr the edge top (s edgetepated 4); (stepspot Raman on 4); the Ramansignals TLC signals wereplate collectedwas were detected collected on the under drugon the the concentrateddrug-concentratedlaser, and the parts micro-Raman (step parts 5); (step finally, imaging 5); ﬁnally, the mapconcentr the was concentratedated obtained spot on(Figure spot the on TLC 2II). the plate TLC platewas detected was detected under under the laser,the laser, and the and micro-Raman the micro-Raman imaging imaging map mapwas obtained was obtained (Figure (Figure 2II).2 II).

I Step 1 Step 2 Step 3 Step 4 Step 5

Figure 2. Cont.

Molecules 2018, 23, 1504 4 of 13 Molecules 2018, 23, x 4 of 13

Molecules 2018, 23, x 4 of 13

Figure 2. The detailed process of spot concentration on TLC plate (I), and micro-Raman imaging map Figure 2. The detailed process of spot concentration on TLC plate (I), and micro-Raman imaging (II). map (II). 2.3. Comparation with TLC-SERS Method 2.3. ComparationFigure 2. with The TLC-SERSdetailed process Method of spot concentration on TLC plate (I), and micro-Raman imaging map (II). TLC-SERS method had been widely used in Raman signal enhancement. In order to compare TLC-SERS method had been widely used in Raman signal enhancement. In order to compare the the two methods, TLC-SERS method was used to enhance Raman signal on TLC plate, in which silver two methods,2.3. Comparation TLC-SERS with TLC-SERS method wasMethod used to enhance Raman signal on TLC plate, in which silver colloids were prepared following the conventional heating method studied by Lee and Meisel [27]. colloids wereTLC-SERS prepared method following had been the widely conventional used in Rama heatingn signal method enhancement. studied byIn Leeorder and to compare Meisel [27]. Taking prednisone acetate as an example, Raman spectra by the TLC-SERS method was compared Takingthe prednisone two methods, acetate TLC-SERS as an example,method was Raman used to spectra enhance by Raman the TLC-SERS signal on methodTLC plate, was in which compared silver with with TLC–SCRS method (Figure 3). We did not see any peak on the TLC spot directly with the TLC–SCRScolloids method were prepared (Figure 3following). We did the not conventional see any peak heating on the method TLC spotstudied directly by Lee with and theMeisel deposition [27]. deposition amount of 10 μg (Figure 3b), while a strong peak at 1658 cm−1 was detected when the amountTaking of 10 prednisoneµg (Figure acetate3b), while as an aexample, strong peakRaman at spectra 1658 cmby the−1 wasTLC-SERS detected method when was the compared TLC-SCRS TLC-SCRS method was used (Figure 3c), which was at the same Raman shift with the reference methodwith was TLC–SCRS used (Figure method3c), which (Figure was 3). atWe the did same not Ramansee any shift peak with on the the TLC reference spot directly powder with (Figure the3 d). powderdeposition (Figure amount 3d). of At 10 the μg same(Figure time, 3b), thewhile other a strong peak speak were at too 1658 small cm−1 towas be detectedseen in thewhen spectra the by the At the same time, the other peaks were too small to be seen in the spectra by the TLC-SCRS method TLC-SCRSTLC-SCRS methodmethod wascompared used (Figure with the3c), spectra which wasof the at thereference same Raman powder. shift The with reason the reference considered was compared with the spectra of the reference powder. The reason considered was that the signal value thatpowder the signal (Figure value 3d). At which the same was time, associated the other with peak thes were deposition too small amountto be seen was in the too spectra small by to the exceed the which was associated with the deposition amount was too small to exceed the noise value. When the noiseTLC-SCRS value. method When compared the TLC–SERS with the methodspectra of was the referenceused, totally powder. different The reason Raman considered shift waspeaks were TLC–SERS method was used, totally different Raman shift peaks were detected. The Raman spectra detected.that the signal The Raman value which spectra was were associated detected with on the the deposition TLC plate amount with was silver too colloids,small to exceed and no the peak was were detectednoise value. on theWhen TLC the plate TLC–SERS with silver method colloids, was us anded, totally no peak different was found Raman (Figure shift 3peaksa). When were the found (Figure 3a). When the TLC–SERS method was used for the prednisone acetate spot on TLC detected. The Raman spectra were detected on the TLC plate with silver colloids, and no peak was TLC–SERS method was used for the prednisone acetate spot− on1 TLC plate, the peaks that arose from −1 plate,found the (Figure peaks 3a). that When arose−1 the from TLC–SERS δas(CH 2method), δ(CH) wa (1395s used cm for) andthe prednisone δ(CH),−1 δ(CC) acetateringD (Figurespot on TLC4) (1012 cm ) δas(CH2), δ(CH) (1395 cm ) and δ(CH), δ(CC)ringD (Figure4) (1012 cm )[28] were strengthened, [28] were strengthened, and there was almost no−1 peak in the position of prednisone− 1acetate’s and thereplate, was the almostpeaks that no arose peak from in the δas(CH position2), δ(CH) of prednisone(1395 cm ) and acetate’s δ(CH), characteristicδ(CC)ringD (Figure peak 4) (1012 (1660 cm cm) −1) characteristic[28] were strengthened, peak (1660 andcm− 1there) (Figure was 3e).almost no peak in the position of prednisone acetate’s (Figure3e). characteristicComparing peak with (1660 TLC–SERS cm−1) (Figure method, 3e). TLC-SCRS method had similar Raman shift with the Comparing with TLC–SERS method, TLC-SCRS method had similar Raman shift with the referenceComparing powder, with although TLC–SERS the signal method, intensity TLC-SCRS was methodweaker. had Furthermore, similar Raman reference shift withsubstances the were reference powder, although the signal intensity was weaker. Furthermore, reference substances were notreference necessary powder, in the although TLC-SCRS the signal method, intensity as thewas Rama weaker.n signalFurthermore, could referencebe compared substances with werethe standard not necessary in the TLC-SCRS method, as the Raman signal could be compared with the standard atlas,not necessary directly. inFrom the TLC-SCRSthis point, method, the TLC-SCRS as the Rama methodn signal was could more be convenient. compared with the standard atlas, directly. From this point, the TLC-SCRS method was more convenient. atlas,Furthermore, directly. From since this point,the target the TLC-SCRS reagent on method TLC sometimeswas more convenient. cannot be observed by SERS, which has Furthermore,Furthermore, since since the targetthe target reagent reagent on on TLC TLC sometimes sometimes cannot be be observed observed by by SERS, SERS, which which has has selectivity via the binding affinity on the metal surface, the TLC-SCRS method is important. selectivityselectivity via the via binding the binding afﬁnity affinity on on the the metal meta surface,l surface, the the TLC-SCRSTLC-SCRS method method is isimportant. important.

15000 15000

e e

1000010000 d d c c 5000 5000 b Relative Intensity/a.u. Relative b Relative Intensity/a.u. Relative a 0 a 0 1000 1500 2000 1000Raman shift( cm -1 ) 1500 2000 Raman shift( cm -1 )

Figure 3. Raman spectra of prednisone acetate (a: detected by TLC-SERS method without prednisone Figure 3. Raman spectra of prednisone acetate (a: detected by TLC-SERS method without prednisone acetate, b: detected directly on TLC spot without prednisone acetate, c: detected by the TLC-SCRS Figureacetate, 3. b Raman: detected spectra directly of onprednisone TLC spot withoutacetate (prednisonea: detected acetate, by TLC-SERS c: detected method by the without TLC-SCRS prednisone method, d: detected directly on reference powder, and e: detected by the TLC-SERS method. acetate,method, b d: :detected detected directly on on reference TLC spot powder, without and prednisone e: detected acetate,by the TLC-SERS c: detected method. by the The TLC-SCRS The deposition amount is 10 µg). method,deposition d: amountdetected is 10directly μg). on reference powder, and e: detected by the TLC-SERS method. The deposition amount is 10 μg).

Molecules 2018, 23, 1504 5 of 13 MoleculesMolecules 2018 2018, ,23 23, ,x x 55 ofof 1313

FigureFigureFigure 4. 4. 4.The The The commoncommon common structure structure of ofof steroids. steroids.steroids.

2.4.2.4.2.4. Raman Raman Raman Spectra Spectra Spectra by by by TLC–SCRS TLC–SCRS TLC–SCRS ReferenceReferenceReference substance substance substance solutions solutions solutions were were were analyzed analyzed analyzed byby by use use of of this thisthis TLC–SCRS TLC–SCRSTLC–SCRS me me methodthodthod by by by acquiring acquiring acquiring the the the spotsspotsspots spectra spectra spectra (Figure (Figure (Figure5) 5) on5) on on TLC TLC TLC plate plate plate in in in Figure Figure Figure1. 1. 1.

40004000 FF E 30003000 E Molecules 2018, 23, x 5 of 13 DD 20002000 CC

10001000 Relative Intensity/a.u. Relative Relative Intensity/a.u. Relative BB

0 0 AA 12001200 1400 1400 1600 1600 1800 1800 2000 2000 RamanRaman shift(cm-1) shift(cm-1)

Figure 4. The common structure of steroids. FigureFigureFigure 5. 5.Raman 5. Raman Raman spectra spectra spectra of of of reference reference reference substancesu substancebstance solutions solutions by byby TLC–SCRS TLC–SCRS (deposition (deposition (deposition amount amount amount of of of10 10 10μ μg).g).µ g). 2.4. Raman Spectra by TLC–SCRS A, ABA, ,CB B,, ,CD C, ,,D ED,, ,E FE,: ,F prednisone,F: :prednisone, prednisone, prednisone prednisone prednisone acetate, acetate, acetate, prednisolone,prednisolo prednisolone,ne, hydrocortisone, hydrocortisone,hydrocortisone, hydrocortisone hydrocortisone hydrocortisone acetate, acetate, acetate, Reference substance solutions were analyzed by use of this TLC–SCRS method by acquiring the andandand dexamethasone. dexamethasone. dexamethasone.spots spectra (Figure 5) on TLC plate in Figure 1.

RamanRaman spectra spectra of 4000of reference reference substance substance prednisone, prednisone, pr prednisoneednisoneF acetate, acetate, prednisolone, prednisolone, hydrocortisone, hydrocortisone, E Raman spectra3000 of reference substance prednisone, prednisone acetate, prednisolone, hydrocortisonehydrocortisone acetate, acetate, and and dexamethasone dexamethasone were were detected detected directly directlyD on on reference reference substance substance powders, powders, hydrocortisone, hydrocortisone2000 acetate, and dexamethasone were detected directly on reference respectivelyrespectively (Figure (Figure S1). S1). C 1000 substance powders, respectively Intensity/a.u. Relative (Figure S1). B AsAs we we can can see see in in 0 Figures Figures 5 5 and and S1, S1, and and Table Table 1, 1, the the Raman RamanA shift shift on on these these spectra spectra by by TLC–SCRS TLC–SCRS 1200 1400 1600 1800 2000 Raman shift(cm-1) methodmethodAs we were were can basically basically see in Figure the the same same5 and as as those those Figure obtained obtained S1, and fr fromom Table reference reference1, the chemicals, Ramanchemicals, shift indicating indicating on these that that spectra we we could could by TLC–SCRS methodFigure were 5. Raman basically spectra of reference the substance same solutions as bythose TLC–SCRS (deposition obtained amount fromof 10 μg). reference chemicals, indicating that obtainobtain the the spectral spectralA, B , character Ccharacter, D, E, F: prednisone, by by prednisone the the TLC–SCRSacetate, TLC–SCRS prednisolone, hydrocortisone, meth method,od, hydrocortisone although although acetate, there there were were differences differences in in the the peak peak weheight,height, could obtainpeak peak separation, separation, theand spectraldexamethasone. and and character shape. shape. by the TLC–SCRS method, although there were differences in Raman spectra of reference substance prednisone, prednisone acetate, prednisolone, hydrocortisone, the peak height,hydrocortisone peak separation, acetate, and dexamethasone and shape. were detected directly on reference substance powders, respectivelyTableTable (Figure 1. 1. Raman Raman S1). spectral spectral characteristic characteristic peaks peaks of of the the six six reference reference substances. substances. As we can see in Figures 5 and S1, and Table 1, the Raman shift on these spectra by TLC–SCRS method were basically the same as those obtained from reference chemicals, indicating that we could Table 1. Raman spectral characteristic peaks of the six reference substances. −1 obtain the spectral character by the TLC–SCRS method, although there were differencesRamanRaman in theShift Shift peak RamanRaman Shift Shift (cm (cm−1) ) AdulterantsAdulterants height, peak separation, and shape.StructureStructure (cm(cm−1−)1 )of of Spot Spot on on ofof Reference Reference IntensityIntensity Raman Shift (cm−1) Raman Shift (cm−1) AdulterantsTable 1. Raman Structure spectral characteristic peaks of the six reference substances. TLCTLC PowderPowder Intensity Raman Shift ofRaman Spot Shift on(cm−1)TLC of Reference Powder OO 16571657 16581658 s s Adulterants Structure (cm−1) of Spot on of Reference Intensity OHOH CH TLC Powder CH3 3 O O OHOH1657 1658 s O OH CH3 Prednisone O OH PrednisonePrednisone Prednisone CHCH3 HH 1657 1658 s CH3 3 H (A)(A)(A) (A) 1602 16071602 16021602 w 160716071607 w w H H Molecules 2018, 23, x 6 of 13 O HH HH 1658 1660 s OO Prednisone acetatePrednisone 165816581658 166016601660 s s acetate 1603 1606 w (B) (B) 1603 1606 w

O 1655 1658 s OH OH CH3 H OH Prednisolone CH3 (C) 1605 1604 w

H H

O 1652 1645 s OH H CH3 HO OH Hydrocortisone CH3 H (D) 1614 1613 s

H H

O 1628 1629 s

O CH3 H CH3 Hydrocortisone HO OH acetate CH3 H 1617 1619 s (E)

H H

O 1661 1658 s

Dexamethasone (F) 1605 1606 w

s, strong; w, weak.

The six glucocorticoids are all steroid hormone drugs and have the same structure of cyclopentanoperhydrophenanthrene (Figure 4). Ring A in the structure has the cyclohexene structure, which causes two strong Raman signals. The signal of rings B, C, and D were weak, so the characteristic peaks all came from ring A. The Raman scattering characteristic peak of hydrocortisone and hydrocortisone acetate were similar (Table 1), and there were double strong peaks between 1652 cm−1 and 1613 cm−1, both coming from the cyclohexene structure, and peak separations were both less than 40 cm−1, so it was difficult to identify hydrocortisone and hydrocortisone acetate only by their Raman spectra. The Raman scattering characteristic peaks of prednisone, prednisone acetate, prednisolone, and dexamethasone acetate were similar, and there were a strong peaks and a weak peaks, both coming from the cyclohexadiene structure. There was still a strong peak between 1655 cm−1 and 1661 cm−1, but due to the influence of 1,2-cyclohexadiene, the signal between 1598 cm−1 and 1603 cm−1 was attenuated to a weak peak. Additionally, peak separations were all more than 50 cm−1. It was also difficult to identify prednisone, prednisone acetate, prednisolone, and dexamethasone only by their Raman spectra. Thus, there were distinct differences between cyclohexene and cyclohexadiene steroid hormone drug structures in terms of their Raman spectra. Only the two types of compounds could be identified by Raman spectroscopy.

Molecules 2018, 23, 1504 6 of 13 Molecules 2018, 23, x 6 of 13

PrednisoneMolecules 2018 , 23, x Table 1. Cont. 6 of 13 MoleculesMolecules 2018 2018, 23,, x23 , x 6 of6 13of 13 acetate 1603 1606 w (B)

Prednisone −1 −1 Raman Shift (cm ) Raman Shift (cm ) AdulterantsPrednisoneacetatePrednisone Structure 1603 1606 w Intensity acetate 1603 1606 w acetate(B) O 1603 1655 of Spot1606 on1658 TLC w s of Reference Powder (B) (B) OH OH CH3 H OH

Prednisolone O 1655 1658 s CH3 OH 1655 1658 s O O 1655 1658 s OH CH3 (C) OH 1605 1604 w H OH OH OH CH3 OH CH3 OH Prednisolone H HH H OH 1655 1658 s Prednisolone CH Prednisolone 3 Prednisolone(C) CH3 1605 1604 w O CH3 (C) (C) 1605 16051604 w 1604 w (C) H H 1605 1604 w

H HO H H 1652 1645 s O OH O O H CH 3 1652 1645 s HO O OH O OH 16521652 16451645 s s H CHO 3 OH Hydrocortisone OH OH CHHO3 HH CH H CH3 3 OH Hydrocortisone(D) HO HO OH 1614 1613 s Hydrocortisone CH3 H 1652 1645 s Hydrocortisone Hydrocortisone(D) CH H 1614 1613 s CH3H 3 H H (D) (D) 16141614 16131613 s s (D) H H 1614 1613 s O H H H H O O O O 1628 1629 s O 1628 1629 s 1628 1629 s O O O 1628 1629 s O O CH3 H CH O O CH3 H CHO 3 Hydrocortisone OH O CH3 Hydrocortisone HOHO OH O CH3 H CH3 H CH3 Hydrocortisone OH Hydrocortisone acetateHydrocortisoneacetate HO HO OH 1628 1629 s acetate CH H CH3 3 H 1617 1619 s acetateacetate 1617 1619 s (E) CH3 H (E) CH3 H 1617 1619 s (E) (E) 1617 16191617 s 1619 s (E) H H H H H H H H O O O O 1661 1658 s 16611661 1661 16581658 1658 s s s

Dexamethasone Dexamethasone 1661 1658 s DexamethasoneDexamethasoneDexamethasone(F) 1605 1606 w (F) (F) 16051605 160616051606 w w 1606 w (F) (F) 1605 1606 w

s, strong; w, weak. s, strong;s, strong; w, weak.w, weak. The six glucocorticoids are all steroids, strong; horms, w,one strong; weak. drugs and w, have weak. the same structure of cyclopentanoperhydrophenanthreneTheThe six sixglucocorticoids glucocorticoids are are all all(Figuresteroid steroid 4).horm hormRingone one Adrugs indrugs the and andstructure have have the hasthe same thesame structurecyclohexene structure of of cyclopentanoperhydrophenanthrene (Figure 4). Ring A in the structure has the cyclohexene Thecyclopentanoperhydrophenanthrenestructure, six whichglucocorticoids causes two strongare allRaman (Figuresteroid signals. 4). horm RingThe signaloneA indrugs ofthe rings structure andB, C, andhave has D werethe weak,cyclohexenesame so structure the of structure,structure, which which causes causes two two strong strong Raman Raman signals. signals. Th eTh signale signal of ringsof rings B, C,B, andC, and D wereD were weak, weak, so theso the The sixcyclopentanoperhydrophenanthrene glucocorticoidscharacteristic peaks all came arefrom ring all(Figure A. steroid 4). Ring A hormone in the structure drugs has the andcyclohexene have the same structure of characteristiccharacteristicThe Raman peaks peaksscattering all allcame came characteristic from from ring ring A. A.peak of hydrocortisone and hydrocortisone acetate were structure, whichThe Raman causes scattering two strong characteristic Raman signals.peak of hyThdrocortisonee signal of ringsand hydrocortisone B, C, and D wereacetate weak, were so the similarThe (Table Raman 1), scatteringand there werecharacteristic double strong peak peaksof hydrocortisone between 1652 and cm −hydrocortisone1 and 1613 cm−1, acetateboth coming were cyclopentanoperhydrophenanthrenesimilar (Table 1), and there were double (Figure strong peaks4 between). Ring 1652 cm− A1 −1 inand 1613 the cm−1 structure−1, both coming has the cyclohexene structure, characteristicsimilarfrom the (Table cyclohexene peaks 1), andall came therestructure, werefrom anddouble ring peak A. strong separations peaks between were both 1652 less cm than and 40 1613cm−1 ,cm so it, bothwas difficultcoming −1 which causes twoThefromto strongfromidentify Ramanthe the cyclohexene hydrocortisonecyclohexene scattering Raman structure, structure, characteristic and signals. andhydrocortisone and peak peak separationspeak separations The acetateof hy were signaldrocortisone onlywere both byboth lesstheir of less than ringsRamanandthan 40 hydrocortisone40cm spectra.cm−1, B,so, itsoC, was Theit was and difficultRaman difficultacetate D werewere weak, so the characteristic similartoscattering (Tabletoidentify identify characteristic1), hydrocortisone andhydrocortisone there peaks were and ofand double hydrocortisoneprednisone, hydrocortisone strong prednisone acetatepeaks acetate betweenonly acetate, only by byprednisolone,their 1652 their Raman cm Raman−1 andspectra. and spectra. 1613 dexamethasone The cm The Raman−1, Ramanboth coming scattering characteristic peaks of prednisone, prednisone acetate, prednisolone, and dexamethasone from scatteringacetatethe cyclohexene were characteristic similar, structure, and peaks there of and prednisone,were peak a strong separations prednisone peaks and wereacetate, a weakboth prednisolone, peaks,less than both and40 coming cmdexamethasone−1, sofrom it wasthe difficult peaks all came fromacetate ring were similar, A. and there were a strong peaks and a weak peaks, both coming from the acetatecyclohexadiene were similar, structure. and There there waswere still a astrong strong peaks peak betweenand a weak 1655 peaks,cm−1 and both 1661 coming cm−1, but from due the to to identifycyclohexadiene hydrocortisone structure. and There hydrocortisone was still a strong acetate peak between only by 1655 their cm−1 − 1 Ramanand 1661 spectra.cm−1 −1, but dueThe to Raman cyclohexadienethe influence of structure.1,2-cyclohexadie There wasne, the still signal a strong between peak between1598 cm− 16551 and cm 1603 and cm −16611 was cm attenuated, but due to to a The Ramanscattering scatteringthe characteristicinfluence of 1,2-cyclohexadie peaks characteristic of prednisone,ne, the signal prednisone between peak 1598 acetate, of cm−1 − hydrocortisone1 and prednisolone, 1603 cm−1 −1 was and attenuated dexamethasone andto a hydrocortisone acetate were theweak influence peak. Additionally, of 1,2-cyclohexadie peak separationsne, the signal were between all more 1598 than cm 50 cm and−1. 1603It was cm also was difficult attenuated to identify to a acetate weakwere peak. similar, Additionally, and there peak were separations a strong were allpeaks more and than 50a weakcm−1 −1. It peaks,was also bothdifficult coming to identify from − 1the −1 similar (Table1),weakprednisone, and peak. there Additionally, prednisone were acetate,peak double separations prednisolone, were strong analld more dexamethasone than peaks 50 cm . onlybetweenIt was by also their difficult Raman 1652 to identifyspectra. cm and 1613 cm , both coming cyclohexadieneprednisone,prednisone, structure.prednisone prednisone Thereacetate, acetate, was prednisolone, prednisolone, still a strong an dan peakdexamethasoned dexamethasone between 1655only only bycm bytheir−1 andtheir Raman 1661Raman cmspectra. spectra.−1, but due to Thus, there were distinct differences between cyclohexene and cyclohexadiene steroid hormone drug − the influenceThus,structuresThus, there there of in were 1,2-cyclohexadieterms were distinct ofdistinct their differences Raman differencesne, spectra. thebetween between signal Only cyclohexene cyclohexene betweenthe two types and 1598 and cyclohexadiene of cm cyclohexadienecompounds−1 and 1603 steroidcould steroid cm be −hormone1 identifiedwas hormone attenuated drug bydrug to a 1 from the cyclohexenestructures structure, in terms of their andRaman spectra. peak Only separations the two types of compounds were could both be identified less than by 40 cm , so it was difficult to weakstructuresRaman peak. Additionally,spectroscopy. in terms of their peak Raman separations spectra. Only were the all two more types than of compounds 50 cm−1. It couldwas also be identified difficult by to identify RamanRaman spectroscopy. spectroscopy. identify hydrocortisoneprednisone, prednisone and hydrocortisoneacetate, prednisolone, an acetated dexamethasone only only by by their their Raman spectra. spectra. The Raman scattering characteristicThus, peaks there were of distinct prednisone, differences between prednisone cyclohexene and acetate, cyclohexadiene prednisolone, steroid hormone drug and dexamethasone acetate structures in terms of their Raman spectra. Only the two types of compounds could be identified by were similar,Raman and spectroscopy. there were a strong peaks and a weak peaks, both coming from the cyclohexadiene structure. There was still a strong peak between 1655 cm−1 and 1661 cm−1, but due to the influence of 1,2-cyclohexadiene, the signal between 1598 cm−1 and 1603 cm−1 was attenuated to a weak peak. Additionally, peak separations were all more than 50 cm−1. It was also difficult to identify prednisone, prednisone acetate, prednisolone, and dexamethasone only by their Raman spectra. Thus, there were distinct differences between cyclohexene and cyclohexadiene steroid hormone drug structures in terms of their Raman spectra. Only the two types of compounds could be identified by Raman spectroscopy. In summary, the combination of the Rf value on the TLC plate and Raman scattering characteristic peaks in the Raman spectra showed complementary advantages, and the specificity of the identification method could be effectively enhanced. For example, the Rf values of prednisolone and hydrocortisone were similar, but there were significant differences in their Raman scattering characteristic peaks. The Raman scattering characteristic peaks of prednisone, prednisone acetate, prednisolone, and dexamethasone were similar, but there were significant differences in the Rf values on the TLC plate. Thus, the specificity of identification was enhanced by the TLC–SCRS method to achieve the purpose of accurate identification.

In summary, the combination of the Rf value on the TLC plate and Raman scattering characteristic peaks in the Raman spectra showed complementary advantages, and the specificity of the identification method could be effectively enhanced. For example, the Rf values of prednisolone and hydrocortisone were similar, but there were significant differences in their Raman scattering characteristic peaks. The Raman scattering characteristic peaks of prednisone, prednisone acetate, prednisolone, and dexamethasone were similar, but there were significant differences in the Rf values on the TLC plate. Thus, the specificity of identification was enhanced by the TLC–SCRS method to achieve the purpose of accurate identification.

2.5. Analysis of Simulated Positive Samples Five kinds of dietary supplements (Table S1) were taken as the negative samples (which were established as not containing prednisone, prednisone acetate, prednisolone, hydrocortisone, Moleculeshydrocortisone2018, 23, 1504 acetate, and dexamethasone by Qiqihar Institute for Food and Drug Control).7 Five of 13 kinds of dietary supplement simulated positive samples were prepared by taking the single oral dose of glucocorticoids as the minimum necessary amount of adulterant in one dosage. Negative sample oral dose of glucocorticoids as the minimum necessary amount of adulterant in one dosage. Negative solution and dietary supplement simulated positive solutions were prepared in accordance with the sample solution and dietary supplement simulated positive solutions were prepared in accordance preparation method of real dietary supplement solutions. The six glucocorticoids in dietary with the preparation method of real dietary supplement solutions. The six glucocorticoids in dietary supplement simulated positive samples were detected, respectively, paying close attention to the supplement simulated positive samples were detected, respectively, paying close attention to the influence of those matrix compositions in dietary supplement simulated positive samples at the same inﬂuence of those matrix compositions in dietary supplement simulated positive samples at the same time. The results showed that the five kinds of substrates in dietary supplements had no interference time. The results showed that the ﬁve kinds of substrates in dietary supplements had no interference on the Raman spectra. on the Raman spectra. Taking dietary supplement 1 as an example of a negative sample, a simulated positive sample Taking dietary supplement 1 as an example of a negative sample, a simulated positive sample solution was prepared (Table S2), and the TLC plate (Figure S2) was obtained by the TLC-SCRS solution was prepared (Table S2), and the TLC plate (Figure S2) was obtained by the TLC-SCRS method. method. The simulated positive sample solution showed spots at the same location with reference The simulated positive sample solution showed spots at the same location with reference substance substance solutions (A, B, C, D, E, F), respectively. However, there were no spots in the simulated solutions (A, B, C, D, E, F), respectively. However, there were no spots in the simulated negative negative samples observed, indicating that the matrix of sample 1 had no interference on the TLC samples observed, indicating that the matrix of sample 1 had no interference on the TLC analysis of analysis of the six glucocorticoids. the six glucocorticoids. Raman spectra of prednisone (A) reference substances and the prednisone spot in the simulated Raman spectra of prednisone (A) reference substances and the prednisone spot in the simulated positive sample were detected (Figure 6). The results showed that the prednisone reference substance positive sample were detected (Figure6). The results showed that the prednisone reference substance and the simulated positive samples had the same Raman scattering characteristic peak, and there was and the simulated positive samples had the same Raman scattering characteristic peak, and there no signal for negative samples. This indicated that the matrix of dietary supplement 1 had no was no signal for negative samples. This indicated that the matrix of dietary supplement 1 had no interference with prednisone’s in situ enrichment Raman spectrum. interference with prednisone’s in situ enrichment Raman spectrum.

3000

2500

2000 c 1500

1000 b

500 Relative Intensity/a.u. Relative 0 a

-500 1500 2000 Ram an shift( cm -1 )

FigureFigure 6.6. TLC-SCRSTLC-SCRS ofof prednisoneprednisone (A)(A) ((aa:: simulatedsimulated negativenegative sample;sample; bb:: simulatedsimulated positivepositive sample;sample; andand cc:: referencereference substancesubstance ofof prednisone).prednisone).

InIn thethe samesame way,way, thethe RamanRaman spectraspectra ofof thethe otherother referencereference substancessubstances andand simulatedsimulated positivepositive samplessamples on on TLC TLC plates plates were were detected detected,, respectively respectively (Figure (Figure S3), the S3), resu thelts resultsshowed showed that the thatother the reference other referencesubstances substances and the simulated and the simulated positive positivesamples samples had the had same the Raman same Raman scattering scattering characteristic characteristic peak, peak,and there and was there no was signal no for signal negative for negative samples. samples.It was indicated It was that indicated the matrix that of the dietary matrix supplement of dietary supplement1 had no interference 1 had no interference with TLC withon-site TLC spot-con on-sitecentrated spot-concentrated Raman spectra Raman of spectra prednisone of prednisone acetate, acetate,prednisolone, prednisolone, hydrocortisone, hydrocortisone, hydrocor hydrocortisonetisone acetate, acetate,or dexamethasone. or dexamethasone.

2.6. Inspection of Limit of Detection (LOD) Reference substance solutions (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) were spotted on the TLC plate separately, and the drug deposition amount of each spot was 2–10 µg, respectively. The spectra by TCL-SCRS were recorded in Figure7. Molecules 2018, 23, x 8 of 13

2.6. Inspection of Limit of Detection (LOD) Reference substance solutions (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) were spotted on the TLC plate separately, and the drug deposition amount of each spot was 2–10 μg, respectively. The spectra by TCL-SCRS were recorded in Figure 7.

Molecules 2018, 23, 1504 8 of 13

B, Prednisone Acetate

A, Prednisone

2500 2500 10ug 10ug 2000 2000 8ug 8ug 1500 1500

6ug 6ug 1000 1000

4ug 500 4ug 500 Relative Intensity/a.u. Relative Relative Intensity/a.u. Relative

0 2ug 0 2ug

1500 2000 1500 2000 -1 Raman shift( cm -1) Raman shift( cm )

C , P red n iso lo n e D , H yd ro co rtiso n e

2500 2500 10ug 2000 10ug 2000 8ug 1500 8ug 1500 6ug 1000 6ug 1000

500 4ug

Relative Intensity/a.u. Relative 500 4ug Relative Intensity/a.u.

0 2ug 0 2ug

1500 2000 1500 2000 -1 Ram an shift( cm ) R am an sh ift( cm -1)

E, Hydrocortisone Acetate F, Dxam ethasone

2500

2500 10ug 2000 10ug 2000

8ug 8ug 1500 1500

6ug 6ug 1000 1000

500 4ug 500 4ug Intensity/a.u. Relative Relative Intensity/a.u. Relative

0 2ug 0 2ug 1500 2000 -1 1500 2000 Ram an shift( cm ) Ram an shift( cm -1)

FigureFigure 7. 7.TheThe Raman Raman spectra spectra of of different different deposition deposition amount amount of of reference reference substances substances on on TLC. TLC.

The LOD analysis of six reference substances was shown in Figure 8. When S/N was 3, the The LOD analysis of six reference substances was shown in Figure8. When S/N was 3, deposition amount of the corresponding reference substances (ug) was the limit of detection (LOD). the deposition amount of the corresponding reference substances (ug) was the limit of detection (LOD). The LOD of reference substances (A, B, C, D, E, and F) were 4 μg, 4 μg, 4 μg, 6 μg, 6 μg, and 4 μg, The LOD of reference substances (A, B, C, D, E, and F) were 4 µg, 4 µg, 4 µg, 6 µg, 6 µg, and 4 µg, lower lower than the deposition amounts which were related with the minimum necessary amount of than the deposition amounts which were related with the minimum necessary amount of adulterants adulterants for drug use (Table S2). Thus, the TLC-SCRS method could be used to detect for drug use (Table S2). Thus, the TLC-SCRS method could be used to detect glucocorticoids illegally glucocorticoids illegally added to dietary supplements. When S/N was more than 8, the signal noise added to dietary supplements. When S/N was more than 8, the signal noise ratio was linear to the ratio was linear to the deposition amount of the corresponding reference substances (ug), and the deposition amount of the corresponding reference substances (ug), and the linear equations were linear equations were y(A) = 4.45x − 24.93 (r = 0.9970), y(B) = 3.94x − 20.19 (r = 0.9909), y(C) = 4.13x − y(A) = 4.45x − 24.93 (r = 0.9970), y(B) = 3.94x − 20.19 (r = 0.9909), y(C) = 4.13x − 22.03 (r = 0.9908), 22.03 (r = 0.9908), y(D) = 4.55x − 33.27 (r = 0.9937), y(E) = 4.34x − 30.29 (r = 0.9919), and y(F) = 3.96x − y(D) = 4.55x − 33.27 (r = 0.9937), y(E) = 4.34x − 30.29 (r = 0.9919), and y(F) = 3.96x − 20.11 (r = 0.9954). 20.11 (r = 0.9954). The recoveries of six glucocorticoids in simulated positive samples were 95.6%, The recoveries of six glucocorticoids in simulated positive samples were 95.6%, 92.3%, 93.5%, 96.1%, 92.3%, 93.5%, 96.1%, 92.7%, and 94.6%, respectively. 92.7%, and 94.6%, respectively.

Molecules 2018, 23, 1504 9 of 13 Molecules 2018, 23, x 9 of 13 Molecules 2018, 23, x 9 of 13

60 60 Prednisone Prednisone Prednisone acetate 50 Prednisone acetate 50 Prednisolone Prednisolone Hydrocortisone Hydrocortisone 40 Hydrocortisone acetate 40 Hydrocortisone acetate Dexamethasone Dexamethasone 30 30

20 ratio Signal-noise Signal-noise ratio Signal-noise 10 10

0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Deposition amount/μg Deposition amount/μg

Figure 8. The LOD analysis of sixsix reference substances. Figure 8. The LOD analysis of six reference substances. 2.7. Detection of Real Samples 2.7.2.7. Detection Detection of ofReal Real Samples Samples Based on the research above, the Rf value on the TLC and the characteristic peaks in the Raman BasedBased on on the the research research above, above, the the R Rf valuef value on on the the TLC TLC and and the the characteristic characteristic peaks peaks in in the the Raman Raman spectra related to the six glucocorticoids had been found, and it was reasonable to take them as the spectraspectra related related to to the the six six glucocorticoids glucocorticoids had had been been fo found,und, and and it itwas was reasonable reasonable to to take take them them as as the the monitoring index for further investigation of adulteration in dietary supplements. In order to further monitoringmonitoring index index for for further further investigation investigation of of adulteration adulteration in in dietary dietary supplements. supplements. In In order order to to further further confirm the performance of TLC-SCRS, it was employed to detect five real samples provided by the confirmconﬁrm the the performance performance of of TLC-SCRS, TLC-SCRS, it itwas was empl employedoyed to to detect detect five ﬁve real real samples samples provided provided by by the the Qiqihar Institute for Food and Drug Control, including decoction, powder, capsule, and tablet. QiqiharQiqihar Institute Institute for for Food Food and and Drug Drug Control, Control, in includingcluding decoction, decoction, powder, powder, capsule, capsule, and and tablet. tablet. There was no spot detected in samples 1, 2, 3, and 4, indicating there were no adulterants ThereThere was was no nospot spot detected detected in samples in samples 1, 2, 3, 1,and 2, 4,3, indicating and 4, there indicating were no there adulterants were no (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and (prednisone,adulterants (prednisone,prednisone prednisoneacetate, prednisolone, acetate, prednisolone, hydrocortisone, hydrocortisone, hydrocortisone hydrocortisone acetate, acetate,and dexamethasone) in the four samples. There was only a spot in sample 5 (Rf = 0.27) that appeared at dexamethasone)and dexamethasone) in the infour the samples. four samples. There There was only was onlya spot a spotin sample in sample 5 (Rf5 = (R 0.27)f =0.27) that thatappeared appeared at almost the same Rf value with prednisolone (Rf = 0.25) or hydrocortisone (Rf = 0.28). We obtained the almostat almost the same the same Rf value Rf value with with prednisolone prednisolone (Rf = (R0.25)f = 0.25)or hydrocortisone or hydrocortisone (Rf = (R0.28).f = 0.28).We obtained We obtained the spectra of the spot in sample 5 after careful inspection, which was compared with the spectra of spectrathe spectra of the of spot the spotin sample in sample 5 after 5 after careful careful inspection, inspection, which which was was compared compared with with the the spectra spectra of of prednisolone and hydrocortisone references. As shown in Figure 9, the Raman spectra indicating the prednisoloneprednisolone and and hydrocortisone hydrocortisone references. references. As As shown shown in in Figure Figure 9,9 ,the the Raman Raman spectra spectra indicating indicating the the characteristic peaks of hydrocortisone were detected in sample 5. There were no illegal ingredients characteristiccharacteristic peaks peaks of of hydrocortisone hydrocortisone were were detected detected in in sample sample 5. 5.There There were were no no illegal illegal ingredients ingredients mentioned above in samples 1, 2, 3, and 4, but hydrocortisone was found in sample 5. mentionedmentioned above above in in samples samples 1, 1,2, 2,3, 3,and and 4, 4,but but hydrocortisone hydrocortisone was was found found in in sample sample 5. 5.

I I

1 2 3 4 5 1 2 3 4 5

Figure 9. Cont.

Molecules 2018, 23, 1504 10 of 13 Molecules 2018, 23, x 10 of 13

A B C D E F 1 2 3 4 5

III

10000

8000

6000 C

4000 D 2000 Ralative Intencity/a.u. Ralative 0 5

-2000 1500 2000 R am an S h ift/(cm -1) FigureFigure 9. 9.Five Five real real samples samples used used in in TLC-SCRS TLC-SCRS detection detection (I ),(I), results results from from TLC TLC analysis analysis of of ﬁve five real real samplessamples developed developed with with dichloromethane-acetone-methanol dichloromethane-acetone-methanol 12:2:0.5 12:2:0.5 (v/ (vv/vv/)(v)II (II),), (A–F: (A–F: prednisone, prednisone, prednisoneprednisone acetate, acetate, prednisolone, prednisolone, hydrocortisone, hydrocortisone, hydrocortisone hydrocortisone acetate, acetate, and and dexamethasone dexamethasone referencesreferences 1–5: 1–5: sample sample 1–5), 1–5), andand the Raman Raman spectra spectra obtained obtained from from prednisolone, prednisolone, hydrocortisone, hydrocortisone, and andsample sample 5 ( 5III (III). ).

2.8.2.8. HPLC-MS HPLC-MS Veriﬁcation Verification ToTo further further conﬁrm confirm the the TLC-SCRS TLC-SCRS results, results, all all the the real real samples samples were were also also analyzed analyzed by by HPLC-MS, HPLC-MS, andand the the same same results results were were obtained obtained (Figure (Figure S4). S4). There There were were no no illegal illegal ingredients ingredients in in samples samples 1, 1, 2, 2, 3, 3, andand 4. 4. However, However, hydrocortisone hydrocortisone was was found found in in sample sample 5. 5. Although Although TLC-SCRS TLC-SCRS was was less less sensitive sensitive than than HPLC-MS,HPLC-MS, the the six six glucocorticoids glucocorticoids could could be be accurately accurately and and quickly quickly detected detected by by use use of of the the TLC-SCRS TLC-SCRS methodmethod at at effective effective doses, doses, which which were were above above the the LOD LOD of of the the TLC-SCRS. TLC-SCRS.

3.3. Experimental Experimental

3.1.3.1. Materials Materials and and Reagents Reagents TLCTLC aluminum aluminum plates plates (Merck (Merck KGaA, KGaA, Darmstadt, Darmstadt, Germany) Germany) consisted consisted of of high-performance high-performance silica silica ± µ ± gelgel 60 60 F254 F254plates, plates, the the silica silica gel gel particle particle size size was was 8 8 ±2 2 m,μm, and and the the layer layer thickness thickness was was 0.2 0.2 ±0.03 0.03 mm. mm. ThereThere was was a ﬂuorescinga fluorescing additive, additive, F254 F254, on, on the the plates plates used used for for the the visualization visualization of of the the spot. spot. An An invisible invisible spotspot in in the the sunlight sunlight could could be be seen seen under under UV UV light ligh oft of 254 254 nm nm because because of of the the ﬂuorescent fluorescent indicators. indicators. TheThe six six reference reference substances substances (prednisone, (prednisone, prednisone prednisone acetate, acetate, prednisolone, prednisolone, hydrocortisone, hydrocortisone, hydrocortisonehydrocortisone acetate, acetate, and and dexamethasone) dexamethasone) were were purchased purchased from from the National the National Institutes Institutes for Food for andFood Drugand Control Drug Control (Beijing, (Beijing, China), China), and were and diluted were diluted to the desired to the desired concentrations concentrations by using by analytical-grade using analytical- Anhydrousgrade Anhydrous ethanol wasethanol purchased was purchased from Tianjin from Tianli Tianji Chemicaln Tianli ReagentChemical Co. Reagent Ltd. (Tianjin, Co. Ltd. China). (Tianjin, China).

Molecules 2018, 23, 1504 11 of 13

3.2. Apparatus Separated spots were located under 254 nm by use of an ultraviolet analyzer (YOKO-2F; Wuhan YOKO Technology Ltd., Wuhan, China). Spot absorption spectra were obtained by use of a DXR™ xi Raman Imaging Microscope (Thermo Fisher Scientiﬁc, Waltham, MA, USA) with an excitation wavelength of 780 nm, a resolution of 5 cm−1 and a 10× long working distance microscope objective. The excitation power was 24 mW, the integration time was 0.2 s, and the number of scans was 20. The scan range was 100–3300 cm−1, and a 50 µm confocal pinhole DXR780 full range grating (400 line/mm) was used. The detector was a TE-cooled electron-multiplying CCD (EMCCD).

3.3. Sample Preparation Reference substance solutions (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) were prepared by dissolving each reference substance in ethanol ultrasonically at a concentration of 1 mg/mL for 10 min, separately. Mixture solutions (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) were prepared by dissolving every reference substance in ethanol ultrasonically at a concentration of 1 mg/mL for 10 min. Simulated negative sample solutions were prepared by mixing single dosage negative sample drugs (which were established to not contain prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone by Qiqihar Institute for Food and Drug Control) with 5 mL ethanol, treating with ultrasound for 10 min, and then filtrating with a microfiltration membrane. Simulated positive sample solutions were prepared by mixing six reference substances (prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone) with the simulated negative sample solutions at a concentration of 1 mg/mL, treating with ultrasound for 10 min, and then filtrating with a microfiltration membrane. Real sample solutions were prepared by grinding or dissolving real dietary supplements and then extracting ultrasonically with ethanol, and then filtrating with microfiltration membrane.

4. Conclusions In summary, a novel combined technique of TLC and SCRS was established to separate and detect small amounts of the six glucocorticoids added to dietary supplements for the ﬁrst time. Additionally, the developed TLC-SCRS method showed enough sensitivity, speciﬁcity, and stability for the ingredients we detected. The optimum SCRS experimental conditions were found. Real samples were then analyzed by use of the TLC-SCRS method and the results were consistent with that obtained by the conventional method of HPLC-MS. The newly-developed TLC-SCRS method without reference substances was much simpler and faster for the detection of adulterants in real dietary supplements than classical methods. We hope the new method may provide more opportunities for the detection of illegal adulterants.

Supplementary Materials: The following are available online, Figure S1: Raman spectra of reference substance powders, Figure S2: TLC of the simulated positive sample, Figure S3: TLC on-site spot concentrated Raman spectra of the simulated positive sample, Figure S4: HPLC-MS results of sample 5, Table S1: The main drug matrix of 5 kinds of dietary supplement, Table S2: The preparation of simulated positive sample solutions. Author Contributions: Conceptualization, L.L. and X.L.; Data curation, X.L.; Formal analysis, X.L.; Funding acquisition, X.L.; Investigation, X.L. and T.X.; Project administration, L.L.; Software, X.L. and F.X.; Supervision, L.L. and X.L.; Validation, X.L.; Visualization, W.D.; Writing—original draft, F.X. Funding: This research was funded by the Fundamental Research Funds for Education Department of Heilongjiang Province grant number [2016-KYYWF-0860]. Acknowledgments: The authors wish to thank Wang Chaozhong from Qiqihar Institute for Food and Drug Control for kindly providing samples and the HPLC-MS results of sample 5 in Figure S4. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2018, 23, 1504 12 of 13

References

1. Knapik, J.J.; Steelman, R.A.; Hoedebecke, S.S.; Austin, K.G.; Farina, E.K.; Lieberman, H.R. Prevalence of Dietary Supplement Use by Athletes: Systematic Review and Meta-Analysis. Sports Med. 2016, 46, 103–123. [CrossRef][PubMed] 2. Phattanawasin, P.; Sotanaphun, U.; Sukwattanasinit, T.; Akkarawaranthorn, J.; Kitchaiya, S. Quanitiatative determination of sibutramine in adulterated herbal slimming formulations by TLC-image analysis method. Forensic Sci. Int. 2012, 219, 96–100. [CrossRef][PubMed] 3. Ko, R.J. Adulterants in Asian patent medicines. N. Engl. J. Med. 1998, 339, 847. [CrossRef][PubMed] 4. Ernst, E. Adulteration of Chinese herbal medicines with synthetic drugs: A systematic review. J. Intern. Med. 2002, 252, 107–113. [CrossRef][PubMed] 5. Huang, W.F.; Wen, K.C.; Hsiao, M.L. Adulteration by synthetic therapeutic substances of traditional Chinese medicines in Taiwan. J. Clin. Pharmacol. 1997, 37, 344–350. [CrossRef][PubMed] 6. Fang, F.; Qi, Y.; Lu, F.; Yang, L. Highly sensitive on-site detection of drugs adulterated in botanical dietary supplements using thin layer chromatography combined with dynamic surface enhanced Raman spectroscopy. Talanta 2016, 146, 351–357. [CrossRef][PubMed] 7. Offerhaus, L.; Dukes, M.N.G.; Smits, H.M. “Herbal” medicines and rheumatoid arthritis. Br. Med. J. 1979, 2, 668. [CrossRef][PubMed] 8. Ahmed, S.; Riaz, M. Quantification of corticosteroids as common adulterants in local drugs by HPLC. Chromatographia 1991, 31, 67–70. [CrossRef] 9. Hughes, J.R.; Higgins, E.M.; Pembroke, A.C. Oral dexamethasone masquerading as a Chinese herbal remedy. Br. J. Dermatol. 1992, 127, 543–544. [CrossRef] 10. Stricht, B.V.; Parvais, O.E.; Vanhaelen-Fastre, R.J.; Vanhaelen, M.H. Remedies may contain cocktail of active drugs. BMJ 1994, 308, 1162. [CrossRef] 11. Ford, A.C.; Bernstein, C.N.; Khan, K.J.; Abreu, M.T.; Marshall, J.K.; Talley, N.J.; Moayyedi, P. Glucocorticosteroid Therapy in Inflammatory Bowel Disease: Systematic Review and Meta-Analysis. Am. J. Gastroenterol. 2011, 106, 590–599. [CrossRef][PubMed] 12. Zhu, Q.; Cao, Y.; Cao, Y.; Chai, Y.; Lu, F. Rapid on-site TLC-SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements. Anal. Bioanal. Chem. 2014, 406, 1877–1884. [CrossRef] [PubMed] 13. Joseph, A.M.; Biggs, T.; Garr, M.; Singh, J.; Lederle, F.A. Stealth steroids. N. Engl. J. Med. 1991, 3, 62. 14. Kaliszewski, P.; Konczak, D.; Cholbinski, P.; Wicka, M.; Michalak, D.; Kwiatkowska, D.; Lewandowska-Pachecka, S.; Namiesnik, J.; Pokrywka, A. Budesonide treatment of professional athletes and anti-doping testing-case studies. Acta Pol. Pharm. 2016, 73, 229–237. [PubMed] 15. Marchei, E.; Pellegrini, M.; Pacifici, R.; Zuccaro, P.; Pichini, S. A rapid and simple procedure for the determination of ephedrine alkaloids in dietary supplements by gas chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 2006, 41, 1633–1641. [CrossRef][PubMed] 16. Marchei, E.; Pichini, S.; Pacifici, R.; Pellegrini, M.; Zuccaro, P. A rapid and simple procedure for the determination of synephrine in dietary supplements by gas chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 2006, 41, 1468–1472. [CrossRef][PubMed] 17. Onal, A. Spectrophotometric and HPLC determinations of anti-diabetic drugs, rosiglitazone maleate and metformin hydrochloride, in pure form and in pharmaceutical preparations. Eur. J. Med. Chem. 2009, 44, 4998–5005. [CrossRef][PubMed] 18. Yao, J.; Shi, Y.Q.; Li, Z.R.; Jin, S.H. Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs. J. Chromatogr. B 2007, 853, 254–259. [CrossRef][PubMed] 19. Lokhnauth, J.K.; Snow, N.H. Solid phase micro-extraction coupled with ion mobility spectrometry for the analysis of ephedrine in urine. J. Sep. Sci. 2005, 28, 612–618. [CrossRef][PubMed] 20. Dillon, J.T.; Aponte, J.C.; Tsai, Y.-J.; Huang, Y. Thin layer chromatography in the separation of unsaturated organic compounds using silver-thiolate chromatographic material. J. Chromatogr. A 2012, 1251, 240–243. [CrossRef][PubMed] 21. Jim, S.; Foroughi-Abari, A.; Krause, K.; Li, P.; Kupsta, M.; Taschuk, M.; Cadien, K.; Brett, M. Ultrathin- layerchromatographynanostruc-tures modified by atomic layer deposition. J. Chromatogr. A 2013, 1299, 118–122. [CrossRef][PubMed] Molecules 2018, 23, 1504 13 of 13

22. Vicario, A.; Sergo, V.; Toffoli, G.; Bonifacio, A. Surface-enhanced Raman spectroscopy of the anti-cancer drug irinotecan in presence of human serum albumin. Colloids Surf. B 2015, 127, 41–46. [CrossRef][PubMed] 23. Huang, R.F.; Han, S.Y.; Li, X. Detection of tobacco-related biomarkers in urine samples by surface-enhanced Raman spectroscopy coupled with thin-layer chromatography. Anal. Bioanal. Chem. 2013, 405, 6815–6822. [CrossRef][PubMed] 24. Freye, C.E.; Crane, N.A.; Kirchner, T.B.; Sepaniak, M.J. Surface enhanced Raman scattering imaging of developed thin-layer chromatography plates. Anal. Chem. 2013, 85, 3991–3998. [CrossRef][PubMed] 25. Pozzi, F.; Shibayama, N.; Leona, M.; Lombardi, J.R. TLC-SERS study of Syrian rue (Peganum harmala) and its main alkaloid constituents. J. Raman Spectrosc. 2013, 44, 102–107. [CrossRef] 26. Wu, S.M.; Chen, S.H.; Wu, H.L. Thin-layer chromatographic detection of glucocorticoids. Gaoxiong Yi Ke Za Zhi 1991, 7, 545–549. 27. Lee, P.; Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem. 1982, 86, 3391–3395. [CrossRef] 28. Dou, W.-H.; Zhou, G.-M.; Kang, Q.-Q. Study of the Epimers of Dexamethasone Sodium Phosphate and Betamethasone Sodium Phosphate by FTIR, FT-Raman and SERS. Spectrosc. Spectr. Anal. 2012, 32, 2664–2668.

Sample Availability: Samples of prednisone, prednisone acetate, prednisolone, hydrocortisone, hydrocortisone acetate, and dexamethasone are available from the authors.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).