Chromtogrphi (2014) 77:1275 1281 DOI 10.1007/s10337-014-2722-9 ORIGINAL Pre Column Derivtiztion HPLC Procedure for the Quntittion of Aluminium Chlorohydrte in Antiperspirnt Crems Using Quercetin s Chromogenic Regent Eleni Klogri Athnsi Vrvresou Spyridon Ppgeorgiou Evggeli Protopp Ionnis Tsknis Alexios Mtiks Irene Pnderi Received: 1 Mrch 2014 / Revised: 9 My 2014 / Accepted: 20 June 2014 / Published online: 10 July 2014 The Author(s) 2014. This rticle is published with open ccess t Springerlink.com Abstrct This rticle describes the development nd vlidtion of selective high-performnce liquid chromtogrphy method tht llows, fter liquid liquid extrction nd pre-column derivtiztion rection with quercetin, the quntifiction of luminium chlorohydrte in ntiperspirnt crems. Chromtogrphic seprtion ws chieved on n XTerr MS C18 nlyticl column (150 3.0 mm i.d., prticle size 5 μm) using mobile phse of cetonitrile:wter (15:85, v/v) contining 0.08 % trifluorocetic cid t flow rte of 0.30 ml min 1. Ultrviolet spectrophotometric detection t 415 nm ws used. The ssy ws liner over concentrtion rnge of 3.7 30.6 μg ml 1 for luminium with limit of quntittion of 3.74 μg ml 1. Qulity control smples (4.4, 17.1 nd 30.6 μg ml 1 ) in five replictes from five different runs of nlysis demonstrted intrssy precision (% coefficient of vrition <3.8 %), interssy precision (% coefficient of vrition <5.4 %) nd n overll ccurcy (% recovery) between 96 nd 101 %. The method ws used to quntify luminium in ntiperspirnt crems contining 11.0, 13.0 nd 16.0 % (w/w) luminium chlorohydrte, respectively. E. Klogri I. Pnderi (*) Division of Phrmceuticl Chemistry, Deprtment of Phrmcy, University of Athens, Pnepistimiopolis, Zogrfou, 157 71 Athens, Greece e-mil: ipnderi@phrm.uo.gr A. Vrvresou S. Ppgeorgiou E. Protopp I. Tsknis Deprtment of Aesthetics nd Cosmetology, The School of Helth nd Cring Professions, Technologicl Eduction Institution of Athens, Ag. Spyridonos str., 122 10 Athens, Greece A. Mtiks Medicl Oncology Deprtment, University Hospitl of Herklion, Minz, Greece Keywords RP-HPLC Pre-column derivtiztion Aluminium chlorohydrte Quercetin Method development Vlidtion Introduction Aluminium slts, such s luminium chlorohydrte tht hs been introduced in the mrket since 1947, re the ctive ingredients of ntiperspirnt in underrm nd bodycre cosmetics pplied to the underrm nd brest re [1]. The effects of widespred, long-term nd incresing use of luminium slts in these cosmetics remin unknown. Aluminium is known to hve genotoxic profile, cpble of cusing both DNA ltertions nd epigenetic effects, nd this could be consistent with potentil role in brest cncer [2]. Results reported in recent reserch demonstrte tht luminium in the form of luminium chloride or luminium chlorohydrte cn interfere with the function of oestrogen receptors of MCF7 humn brest cncer cells, both in terms of lignd binding nd in terms of oestrogen-regulted reporter gene expression [3]. This dds luminium to the incresing list of metls cpble of interfering with oestrogen ction nd termed metlloestrogens [4]. The use of luminium ntiperspirnts hs lso been linked with incresed risk of Alzheimer s disese due to the possible systemic ccumultion of luminium; however this hypothesis remins controversil [5 7]. Although much reserch hs been undertken into the ntiperspirnt properties of number of luminium slts, very little of this work is focused upon the quntifiction of these products in cosmetic formultions. Given tht the toxicity of luminium hs been widely recognized, reducing the concentrtion of this metl in ntiperspirnts is mtter of urgency nd there is rel need to set up nlyticl methods to quntitte luminium slts in underrm cosmetics.
1276 E. Klogri et l. Hyphented techniques by combining vrious chromtogrphic techniques with tomic spectrometry/mss spectrometry re the most efficient for the determintion of luminium in humn body fluids [8, 9]. Recently, high-performnce liquid chromtogrphy interfced to flme tomic spectrometry (HPLC FAAS) [10] nd post-column derivtiztion procedure with 4,5-dihydroxy-1,3 benzene disulfonic cid disodium slt [11] hve been used to quntify luminium fluoride complexes in groundwter smples. Severl high-performnce liquid chromtogrphy methods hve been reported for the nlysis of luminium in vrious mtrixes (queous, serum nd wine smples), the mjority of which include pre-column derivtiztion with lumogllion [12 14], morin [15], quercetin [16], N-o-vnillidine-2-mino-p-cresol [17] nd 8-hydroxyquinoline [18, 19]. Flow nd sequentil injection methods hve been reported for the spectrofluorimetric determintion of luminium in phrmceuticl formultions using chromotropic cid s chromogenic regent [20]. Recently, the binding sites of quercetin with the Al 3+ ion hve been identified using solid-stte NMR [21]. To the best of our knowledge, no methodology hs been described in the literture to quntify luminium slts in cosmetics. Thus, the principl im of this work ws to optimize nd vlidte n nlyticl procedure for the quntittive determintion of luminium chlorohydrte in ntiperspirnt crem smples bsed on pre-column derivtiztion procedure using quercetin, 2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychromen-4-one, s the derivtiztion regent. The method is the first reported ppliction nd could be used for routine nlysis of ntiperspirnt crems contining luminium chlorohydrte, s it complies well with the vlidtion requirements in the cosmetic industries [22]. Experimentl Mterils nd Regents All solvents were of HPLC grde nd were purchsed from Merck (Drmstdt, Germny). Trifluorocetic cid nd mmonium cette of nlyticl regent grde were obtined from Merck (Drmstdt, Germny). Wter ws deionized nd further purified by mens of Milli-Q Plus Wter Purifiction System, Millipore Ltd. Aluminium chlorohydrte solution 50 % (w/w) nd quercetin were purchsed from Sigm-Aldrich (Steinheim, Germny). Cosmetic ntiperspirnt crems contining 11, 13 nd 16 % (w/w) luminium chlorohydrte were produced in the Deprtment of Aesthetics nd Cosmetology of the Technologicl Eductionl Institution of Athens, Greece. The excipients present in crems were: distrch phosphte, llntoin, cetereth-12, cetereth-20, glyceryl sterte, cetyl lcohol, octyl sterte, dimethicone, triethyl citrte, methyl prben, ethyl prben, propyl prben, cyclomethicone, PPG-25-lureth-25, prfum nd qu. Instrumenttion The chromtogrphic equipment used consisted of Spectr Series P100 isocrtic pump (SP ThermoSeprtion, UK) nd Rheodyne Model 7725i injector (Rheodyne Cliforni, CA, USA) with 20 μl loop. The detection ws performed using Wters 486 UV Vis detector (Wters, Milford, MA, USA) operted t 415 nm. Dt cquisition nd nlysis were performed using Empower softwre (Wters, Milford, MA, USA). All glsswre continers were crefully treted with 2.0 M nitric cid for more thn 48 h nd rinsed with wter. Liquid Chromtogrphic Conditions Chromtogrphy ws performed t mbient conditions on n XTerrMS C18 reversed HPLC nlyticl column (150.0 3.0 mm i.d., 5 μm prticle size), Wters (Milford, MA, USA). The mobile phse consisted of cetonitrile: wter (15:85, v/v) contining 0.08 % trifluorocetic cid. It ws filtered through 0.45 μm nylon-membrne filter, GelmnSciences (Northmpton, UK), nd degssed under vcuum prior to use. A flow rte of 0.30 ml min 1 with column inlet pressure of 1,450 psi ws used to seprte the excess of quercetin from the luminium quercetin complex. Chromtogrphy ws performed t 25 ± 2 C with chromtogrphic run time of <7.0 min. Stock nd Working Stndrd Solutions Stock stndrd solution of luminium chlorohydrte, contining 875.0 μg ml 1 luminium, ws prepred by pproprite dilution of the luminium chlorohydrte solution 50 % (w/w) in wter. Stock stndrd solution of the regent, quercetin, 500.0 μg ml 1, ws prepred by dissolving the pproprite mount of the compound in methnol. These solutions were stored in the drk under refrigertion nd were found to be stble for period of 4 weeks. A working stndrd solution of luminium chlorohydrte, contining 43.75 μg ml 1 of luminium, ws prepred by subsequent dilution of the bove-mentioned stock stndrd solution in wter. The working stndrd solution ws freshly prepred every week nd stored in the drk t 4 C. Clibrtion Spiked Crem Smples nd Qulity Control Smple Preprtion Clibrtion spiked crem smples were freshly prepred every working dy t the concentrtion levels of 3.7, 4.4, 6.6, 10.9, 15.3, 17.1, 19.7, 26.2 nd 30.6 μg ml 1 for luminium by ddition of the pproprite liquot of the
Pre-column Derivtiztion HPLC Procedure bove-mentioned working stndrd solution of the nlyte to 25 mg of plcebo crem smples. Qulity control (QC) smples were prepred independently, in n nlogous mnner s the clibrtion spiked crem smples, using seprte stock stndrd solution of the nlyte. QC smples were prepred in plcebo crem t three concentrtion levels (4.4, 17.1 nd 30.6 μg ml 1 ) for luminium. Smple Preprtion nd Derivtiztion Procedure Extrction, clenup nd derivtiztion procedures for crem smples were crried out ccording to the following steps. Exctly, 25 mg of crem smple ws trnsferred into 20 ml volumetric flsk with 2 ml of cetonitrile nd diluted to volume with HCl 0.01 M. The mixture is shken for 2 min nd 2 ml portion of this solution is centrifuged t 18,000 g for 20 min. In 1 ml of the queous phse (bottom lyer) 500 μl of tert-butyl methyl ether is dded nd the mixture is vigorously mixed on vortex mixer for 2 min nd centrifuged t 18,000 g for 15 min. The orgnic lyer is discrded nd 100 μl liquot of this solution is treted with 500 μl of 1.0 M mmonium cette cetic cid buffer (ph 4.5) nd 1.0 ml of 500.0 μg ml 1 quercetin solution. The solution is vigorously mixed on vortex mixer for 1 min nd diluted to 10.0 ml with mixture of cetonitrile methnol (10:90, v/v). A 20 μl liquot is then injected into the chromtogrphic system. Vlidtion Procedure To evlute the linerity of the proposed method, the clibrtion spiked crem smples were prepred nd nlysed in duplicte on three different nlyticl runs. Quntittion ws performed using the pek re of luminium quercetin complex. QC smples were processed in five replictes t ech concentrtion (4.4, 17.1 nd 30.6 μg ml 1 ) for five different nlyticl runs to evlute the intr- nd interssy ccurcy nd precision. The stndrd ddition method ws used to evlute the effect of the excipients on the determintion of luminium chlorohydrte. Thus, six equl mounts of crem smples equivlent to 0.350 mg of luminium chlorohydrte (0.044 mg of luminium) were spiked with different mounts of the working stndrd solution of the nlyte. The spiked crem smples were then nlysed s mentioned in the ssy procedure. Results nd Discussion Optimiztion of the Pre Column Rection Procedure Quercetin (Fig. 1) possesses two possible chelting sites, 3-hydroxy-4-oxo nd 5-hydroxy-4-oxo systems. Literture HO OH HO Fig. 1 Chemicl structure of quercetin Pek re signl bpek re signl 1277 survey revels tht luminium is bonded to 3-hydroxy-4- oxo system nd forms predominntly 1:1 complex with quercetin. There is no evidence tht 3,4 dihydroxyflvone O O OH OH 180000 160000 140000 120000 100000 80000 60000 40000 20000 0 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 Stoichiometric rtio of quercetin: luminium (M/M) cpek re signl 450000.0 400000.0 350000.0 300000.0 250000.0 200000.0 150000.0 100000.0 50000.0 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 ph 400000.0 350000.0 300000.0 250000.0 200000.0 150000.0 100000.0 50000.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Ammonium cette (mm) Fig. 2 Optimiztion of the derivtiztion rection. Plots of the signl of the quercetin luminium complex versus the stoichiometric rtio of quercetin:luminium, M/M, b plots of the signl of the quercetin luminium complex versus the concentrtion of mmonium cette (mm) in the rection medium nd c plots of the signl of the signl of the luminium quercetin complex versus the ph vlue of the rection medium
1278 E. Klogri et l. Fig. 3 Representtive RP-HPLC chromtogrms obtined from the nlysis of blnk crem mtrix smple. b A clibrtion spiked crem smple contining 19.7 μg ml 1 luminium nd c crem smple contining 17.0 μg ml 1 luminium. Chromtogrphic conditions: RP-HPLC on n XTerrMS C18 nlyticl column; mobile phse: cetonitrile:wter (15:85, v/v) contining 0.08 % trifluorocetic cid; flow rte 0.30 ml min 1 nd UV detector t 415 nm forms complex in cid solution [16, 23]. The pplicbility of quercetin s chromogenic regent for the nlysis of luminium in ntiperspirnt crems contining luminium chlorohydrte ws thoroughly investigted. In every step of the optimiztion procedure ll the contributing fctors but one remin constnt nd the optimized vlue is used for the next experiment. The effect of the stoichiometric rtio of quercetin:luminium, M/M on the pek re signl of quercetin luminium complex ws exmined over the rnge of 5:1 300:1 M/M. The results indicted tht once the molr proportion of quercetin:luminium exceeds 20:1 M/M, the pek re of the complex does not significntly increse, remins constnt up to 200:1 (Fig. 2), nd covers the rnge of the clibrtion curve. The effects of the concentrtion (0.5 3.0 M) nd ph (3.0 6.5) of the mmonium cette cetic cid buffer solution tht ws used s the rection solvent were lso investigted. The complex formtion reched the mximum rection yield in 1.0 M mmonium cette cetic cid buffer solution, wheres further increses in the concentrtion of mmonium cette decresed the pek re of the complex (Fig. 2b). Also, the ph of the buffer solution is criticl fctor for the complex formtion. The mximum rection yield ws chieved t ph 4.5, wheres t ph vlues greter thn 5.5 the signl decresed rpidly (Fig. 2c). This effect cn be ttributed to the fct tht under cidic queous solutions, the luminium ion exists minly s Al 3+ nd n increse in ph results in the formtion of complexes of luminium with hydroxide nd finlly the formtion of insoluble luminium hydroxide t neutrl ph. The optimum conditions were the following: rtio of quercetin:luminium greter thn 20:1, M/M in 1.0 M mmonium cette cetic cid buffer solution ph 4.5. The complex ws found to be stble for 80 min prior to the injection into the HPLC system, which is dequte time for the chromtogrphy. Optimiztion of Chromtogrphic Conditions Chromtogrphy ws performed using n XTerr MS C18 (150.0 3.0 mm i.d., 5 μm prticle size) column, while chromtogrphic conditions were optimized to seprte
Pre-column Derivtiztion HPLC Procedure 1279 Tble 1 Anlyticl concentrtion prmeters of the clibrtion equtions for the determintion of luminium, by pre-column derivtiztion HPLC method Medium Concentrtion rnge (μg ml 1 ) Regression eqution r b c Stndrd devition S r slope intercept α/s α d Wter smples Run.7 30.6 S Al = 10301 C Al + 2466 0.998 177 3,152 2,126 1.27 Spiked crem smples Run.7 30.6 S Al = 9750 C Al + 2092 0.998 198 3,651 2,780 0.57 Run 2 3.7 30.6 S Al = 9762 C Al + 68 0.998 119 2,203 2,884 0.03 Run 3 3.7 30.6 S Al = 9756 C Al 1659 0.997 119 2,188 2,865 0.76 Men of three clibrtion curves over period of 1 month Spiked crem smples 3.7 30.6 S Al = 9756 C Al + 167 >0.997 6.1 1,877 <2,780 <0.76 Pek re signl of luminium, S Al, versus the corresponding concentrtion of luminium, C Al b Correltion coefficient c Stndrd error of the estimte d Theoreticl vlue of t t P = 0.05 nd f = n 2 = 6 df, 2.45 quercetin luminium complex from the excess of quercetin nd the crem mtrix excipients. Methnol nd cetonitrile were tried s orgnic modifiers in the mobile phse in combintion with wter. In the present study, cetonitrile ws preferred to methnol s it gve better pek shpe. It ws found tht n increse in the content of cetonitrile s orgnic modifier in the mobile phse could improve pek shpe, wheres n increse in wter content brodened the pek. An increse in the retention of the complex is observed with incresing trifluorocetic cid from 0.02 to 0.12 % (v/v) nd improved pek shpe. Thus, mobile phse consisting of 15 % cetonitrile in wter contining 0.08 % trifluorocetic cid ws used s the optimum. Ech chromtogrphic run ws completed within 7.0 min. The selectivity of the proposed chromtogrphic procedure is illustrted in Fig. 3 with representtive HPLC chromtogrm obtined from the nlysis of plcebo crem smple without the ddition of luminium chlorohydrte (Fig. 3) long with clibrtion spiked crem smple contining 19.7 μg ml 1 of luminium (Fig. 3b) nd chromtogrm obtined from the nlysis of crem smple contining 17.00 μg ml 1 luminium (Fig. 3c). Under the current chromtogrphic conditions, complete seprtion mong the luminium quercetin complex nd the excipients is chieved nd the complex is eluted t 6.07 min. Sttisticl Anlysis of Dt Clibrtion spiked crem smples of luminium chlorohydrte were nlysed in triplicte in three nlyticl runs for the clibrtion procedure. Liner reltionships between the pek re signls of luminium quercetin complex nd the Tble 2 Accurcy nd precision evlution of qulity control smples for luminium (n = 5 runs, five replictes per run) Compound Concentrtion (μg ml 1 ) Aluminium dded 4.4 17.0.6 concentrtion Run 1 (men ± SD) 4.18 ± 0.15 17.06 ± 0.10.28 ± 0.31 Run 2 (men ± SD) 4.08 ± 0.25 17.41 ± 0.23 30.38 ± 0.25 Run 3 (men ± SD) 4.22 ± 0.08 17.32 ± 0.13 30.36 ± 0.29 Run 4 (men ± SD) 4.24 ± 0.13 17.39 ± 0.10.48 ± 0.19 Run 5 (men ± SD) 4.38 ± 0.11 17.34 ± 0.17 30.51 ± 0.23 Overll men 4.22 17.30.40 Intr-ssy RSD (%) 3.7 0.9 0.8 Inter-ssy RSD (%) 5.5 1.8 0.5 % Recovery b 96 101 99 Coefficient of vrition; intr- nd inter-ssy RSDs were clculted by ANOVA b % Recovery = [(overll men ssyed concentrtion 100)/(dded concentrtion)] corresponding concentrtions of luminium were observed s shown by the results presented in Tble 1; the correltion coefficient ws greter thn 0.997. Bck-clculted concentrtions in the clibrtion curves were <3.4 % of the nominl, which re in greement with interntionl guidelines. The insignificnce of intercepts tht ws proven by Student s t test indictes tht there is no effect from the crem s excipients. The limit of detection, LOD, nd the limit of quntifiction, LOQ, for luminium were determined ccording to the definitions of ICH Topic Q2B [22]. In prticulr, the LOD ws clculted using the eqution LOD = 3.3 S/b, nd it ws found to be t the level of 1.24 μg ml 1 while the limit of quntifiction, LOQ, ws ttined using the
1280 E. Klogri et l. Tble 3 Robustness evlution of the pre-column derivtiztion HPLC method for the determintion of luminium chlorohydrte in ntiperspirnt crems Chromtogrphic chnges Prmeters Mesured responses t r b k c T d Concentrtion of luminium chlorohydrte % (w/w) A wvelength of UV detection (414 420 nm) Men (%RSD) 5.07 (0.4) 1.31 (0.8) 1.23 (0.3) 11.3 (2.5) B % trifluorocetic cid in the mobile phse (0.75 0.85 % v/v) Men (%RSD) 5.14 (0.9) 1.29 (1.7) 1.28(0.5) 11.4 (3.3) C % of cetonitrile in the mobile phse (69 71 % v/v) Men (%RSD) 5.12 (2.1) 1.32 (3.4) 1.24 (0.7) 11.5 (3.5) Three prmeters (A, B nd C) were slightly chnged t three levels (1, 0, 1); ech time prmeter ws chnged from level (0), the others remined t level (0) b retention time c cpcity fctor d tiling fctor Tble 4 Quntifiction of luminium chlorohydrte in ntiperspirnt crems by pre-column derivtiztion HPLC method Lot no. % Lbel clim in luminium chlorohydrte Experimentl luminium chlorohydrte concentrtion/100 mg crem men vlue ± SD (n=10) % Recovery % E r 1 11 10.6 ± 0.6 96 ± 5 3.2 2 13 12.7 ± 0.5 97 ± 4 2.6 3 16 15.4 ± 0.6 96 ± 4 3.9 eqution LOQ = 10 S/b (where b is the slope nd S is the stndrd devition of the intercept,, of the regression line) nd it ws found to be t the level of 3.74 μg ml 1. One-wy nlysis of vrince ws used to evlute the intr- nd inter-ssy precision. Results presented in Tble 2 indicte tht intr-ssy reltive stndrd devition vlues, %RSD, were between 0.8 nd 3.7 % for the nlyte, while the inter-ssy %RSD ws no more thn 5.5 %. The overll ssy ws ssessed by % recovery which rnged from 96 to 101 %. A number of orgnic solvents such s hexne, diethyl ether nd ethyl cette were tested for the liquid extrction procedure nd led to poor recoveries. Tert-butyl methyl ether ws finlly chosen s the optimum extrction solvent. The recovery of the extrction procedure ws evluted by compring the slope of the regression eqution obtined from the nlysis of clibrtion spiked crem smples over the slope of the regression eqution obtined from the nlysis of clibrtion smples prepred in wter solution nd nlysed immeditely without smple preprtion procedure (Tble 1). The dt, under the optimum extrction conditions, indicte recovery of 95 % for luminium. To verify the robustness of the method, smll deliberte vritions were introduced round the optiml conditions nd the influence of these vritions in the retention time, cpcity fctor, tiling fctor nd concentrtion of luminium in crem smples ws thoroughly investigted. The prmeters selected to exmine were the percentges of cetonitrile nd trifluorocetic cid in the mobile phse nd the wvelength of UV detection. Replicte injections (n = 3) of crem smple contining 11 % w/w luminium chlorohydrte nd processed ccording to the smple preprtion procedure were performed under smll chnges of the forementioned prmeters. The evlution of the method robustness (Tbles 3, 4) indictes tht there is no significnt difference in the mesured responses fter smll vritions of the selected prmeters. Appliction of the Method to the Anlysis of Rel Smples The proposed method ws evluted in the ssy of three different lots of ntiperspirnt crems contining 11, 13 nd 16 % (w/w) of luminium chlorohydrte, nd the percent lbel clims for luminium chlorohydrte were found to be 97 ± 5, 97 ± 4 nd 96 ± 4, respectively. To further ssess the specificity of the proposed method, recovery studies were lso performed by spiking crem smples with known nd different mounts of luminium chlorohydrte. The regression line of the instrumentl response versus the dded concentrtion of luminium is plotted nd the negtive intercept on the concentrtion xis (x-xis) corresponds to the concentrtion of the nlyte in the crem smple. This vlue is given by the rtio of the intercept nd the slope of the regression line [24], which were found to be 36,890 ± 7,817 nd 17,853 ± 423,
Pre-column Derivtiztion HPLC Procedure respectively. The lbel clim for luminium chlorohydrte using the stndrd ddition method ws found to be 10.4 % w/w. Conclusions Aluminium chlorohydrte is the ctive ingredient of ntiperspirnt in underrm nd bodycre cosmetics pplied to the underrm nd brest re. No methodology hs been previously described to quntitte luminium chlorohydrte in ntiperspirnt crems. The proposed pre-column derivtiztion HPLC method using quercetin s chromogenic regent ws evluted over the linerity, precision, ccurcy nd specificity nd proved to be convenient nd effective for the determintion of luminium chlorohydrte in crems. Acknowledgments This work ws supported by the Technologicl Eductionl Institution of Athens through Archimedes III ction. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution License which permits ny use, distribution, nd reproduction in ny medium, provided the originl uthor(s) nd the source re credited. References 1. Lden K (1988) Felger CB Antiperspirnts nd Deodornts: Cosmetic Science nd Technology Series, vol 7. Mrcel Dekker, New York 2. Drbre PD, Mnnello F, Exley C (2013) J Inorg Biochem 128:257 261 1281 3. Scheel C, Weinberg RA (2012) Semin Cncer Biol 22:396 403 4. Drbre PD (2006) J Appl Toxicol 26:191 197 5. Exley C (1998) Mol Med Tody 4:107 109 6. Pohnk M (2014) Environ Toxicol Phrmcol 37:455 459 7. Di Lorenzo F, Di Lorenzo B (2013) Neuro Endocrinol Lett 34:504 507 8. Chen B, Zeng Y, Hu B (2010) Tlnt 81:180 186 9. Murko S, Milcic R, Krlj B, Scncr J (2009) Anl Chem 81:4929 4936 10. Frnkowski M, Zioł-Frnkowsk A, Siepk J (2010) Tlnt 80:2120 2126 11. Frnkowski M (2012) Microchemicl J 101:80 86 12. Wu J, Zhou CY, Chi H, Wong MK, Lee HK, Ong HY, Ong CN (1995) J Chromtogr B Biomed Appl 663:247 253 13. Ren JL, Zhng J, Luo JQ, Pei XK, Jing ZX (2001) Anlyst 126:698 702 14. Lee BL, Chu LH, Ong HY, Yng HG, Wu J, Ong CN (1996) Clin Chem 42:1405 1411 15. Lin HZ, Kng YF, Ysin A, Bi SP, Sho DL, Chen YJ, Di LM, Tin LC (2003) J Chromtogr A 993:179 185 16. Lin H, Kng Y, Bi S, Askin Y, Sho D, Li D, Chen Y, Di L, Gn N, Tin L (2004) Tlnt 62:43 50 17. Kr D, Fisher A, Hill SJ (2008) Anl Chim Act 611:62 67 18. Kelly MT, Blise A (2006) J Chromtogr A 1134:74 80 19. Kshimur K, Mizushim Y, Hoshino E, Mtsubr S (2003) J Chromtogr B 791:13 19 20. Themelis DG, Kik FS (2006) J Phrm Biomed Anl 41:1179 1185 21. Ahmedov A, Prdowsk K, Wwer I (2012) J Inorg Biochem 110:27 35 22. Interntionl Conference on Hrmonistion (ICH) (1996) Triprtite guideline vlidtion of nlyticl procedures: text nd methodology Q2 (R1): current step 4 version prent guideline dted 27 October 1994, (Complementry Guideline on Methodology dted 6 November 1996 incorported in November 2005) 23. Cornrd JP, Dngleterre L, Lpouge C (2005) J Phys Chem A 109:1044 1051 24. Miller JN (2005) Miller JC Sttistics nd Chemometrics for Anlyticl Chemistry, 5th edn. Person Eduction Ltd, Hrlow