STRATEGIES OF SUNSCREEN SEPARATION BY THIN LAYER CHROMATOGRAPHY

Act Polonie Phrmceutic ñ Drug Reserch, Vol. 69 No. 5 pp. 791ñ797, 2012 ISSN 0001-6837 Polish Phrmceuticl Society ANALYSIS STRATEGIES OF SUNSCREEN SEPARATION BY THIN LAYER CHROMATOGRAPHY ANNA W. SOBA SKA* nd ELØBIETA BRZEZI SKA Deprtment of Anlyticl Chemistry, Chir of Medicl Chemistry, 90-151 Ûdü, MuszyÒskiego 1, Polnd Abstrct: So fr sunscreens seprtion hs been chieved primrily by HPLC or TLC on RP-18 or (less frequently) unmodified silic gel s sttionry phse. The conditions of chromtogrphic seprtion published so fr re not suitble for mny combintions of UV filters found commonly in cosmetic products. It is therefore resonble to seek lterntive conditions tht would mke it possible to seprte these substnces more effectively. Chromtogrphic seprtion of 15 UV filters used commonly for skin nd hir protection ws investigted. The effectiveness of seprtion of investigted compounds by norml- nd reversed-phse thin lyer chromtogrphy ws compred on RP-18, RP-2 nd silic gel 60 sttionry phses. In the cse of some typicl combintions of sunscreens norml-phse chromtogrphy ws found superior compred to the reversed-phse technique proposed in some published ppers. It ws suggested tht sunscreens of totlly different lipophilicity could be seprted by multiple development TLC. Keywords: sunscreens, thin lyer chromtogrphy, seprtion It ppers from the literture survey tht sunscreens in cosmetic products my be quntified by mny nlyticl techniques: UV/VIS spectrophotometry, Rmn spectrometry, column chromtogrphy (HPLC or GC) with vrious detectors (UV, DAD, MS), electrochromtogrphic techniques (MEEKC, MEKC, CE) nd electronlyticl techniques (polrogrphy nd voltmperometry) s well s injection flow methods (1, 2). One of the chromtogrphic techniques used to nlyze sunscreen products is thin lyer chromtogrphy (TLC). Identifiction of UV filters in cosmetic preprtions ws chieved by comprison of R f vlues clculted for the product components with those of the stndrds s well s by spectroscopic techniques (UV, IR, 1 H NMR) fter the chromtogrphic seprtion of nlyzed smples (3, 4). Quntittive nlysis consisted t first in chromtogrphic seprtion, scrpping off the sorbent from chromtogrphic pltes, extrction of the sunscreen from the sorbent with suitble solvent nd spectrophotometric quntifiction of the extrcted substnce (5). Such procedure ws lter replced with much less tedious densitometric evlution of spot chromtogrms. Sherm nd coworkers published series of ppers deling with densitometric nlysis of some sunscreens (octyl 4- methoxycinnmte (6), octyl slicylte (7), benzophenone-3 (8), octyl p-dimethylminobenzote (9) nd octocrylene (10)) on RP-18 sttionry phse with methnol, tetrhydrofurne nd wter (50:35:15, v/v/v) s mobile phse, with UV bsorption detection. TLC ws used to study photodegrdtion of UV filters; chromtogrphic seprtion ws crried out on LiChrospher Si 60F 254s sttionry phse with t-butylmethyl ether nd n-hexne by utomted multiple development TLC. Sunscreen products were compred before nd fter the exposition to UV rys. Bioluminescent detection ws lso used (in the cse of filters nd their degrdtion products exhibiting cytotoxic ctivity bioluminescence of Vibrio fischeri bcteri ws inhibited) (11). In the cse of sunscreen products often contining combintions of severl UV filters of different lipophilicity it ws considered resonble to propose other conditions of chromtogrphic seprtion. The objective of this work ws systemtic review of possibilities offered by vrious sttionry phses (silic gel 60, RP-2 nd RP-18) nd mobile phses with respect to the chromtogrphic seprtion of UV filters most common in the EU (Fig. 1). * Corresponding uthor: e-mil:.sob@poczt.onet.pl 791

792 ANNA W. SOBA SKA nd ELØBIETA BRZEZI SKA Figure 1. Structurl formuls of sunscreens investigted in the course of this study EXPERIMENTAL Mterils nd instrumenttion Solvents Diethyl ether, cyclohexne, ethyl cette, isopropnol, cetic cid, mmoni (25% w/w) ñ POCh Gliwice; tetrhydrofurne, cetonitrile, methnol, cetone, citrte-phosphte buffer ph 3.0, dimethylformmide ñ Chempur; toluene ñ Lch-Ner; dichloromethne, chloroform, n-hexne ñ Fluk; 1,4-dioxne ñ Merck. All solvents were of nlyticl grde. UV filters Neo Heliopn AV (2-ethylhexyl-4- methoxycinnmte) ñ OMC ñ Symrise Eusolex OCR (2-ethylhexyl-2-cyno-3,3- diphenylcrylte) ñ OCR ñ Merck Eusolex 9020 (4-tert-butyl-4í-methoxydibenzoylmethne) ñ AVO ñ Merck Eusolex 6300 (3-(4í-methylbenzylidene)cmphor) ñ MBC ñ Merck Eusolex HMS (3,3,5- trimethylcyclohexyl slicylte) ñ HMS ñ Merck Uvinul T 150 (2,4,6-triniline-(p-crbo-2í-ethylhexyl-1í-oxy)-1,3,5-trizine) ñ ET ñ BASF Eusolex 232 (2-phenylbenzimidzol-5-sulfonic cid) ñ PBS ñ Merck Uvinul M40 (2-hydroxy-4-methoxybenzophenone) ñ BZ3 ñ BASF Neo Heliopn E 1000 (3-methylbutyl-4- methoxycinnmte) ñ IMC ñ Symrise Uvinul A Plus (2-(4-diethylmino-2-hydroxybenzoyl)benzoic cid hexylester) ñ DHHB ñ BASF Uvinul MS40 (2-hydroxy-4-methoxybenzophenone-5-sulfonic cid) ñ BZ4 ñ BASF Eusolex OS (2-ethylhexylslicylte) ñ OS ñ Merck Eusolex 6007 (2-ethylhexyl-4-N,N-dimethylminobenzote) ñ ODP ñ Merck Neo Heliopn AP (disodium 2,2í-(1,4-phenylene)bis(6-sulfo-1H-benzimidzole-4-sulfonte)) ñ PDT ñ Symrise 4-minobenzoic cid ñ PABA ñ Merck All sunscreens were of cosmetic qulity. Chromtogrphic pltes Merck, silic gel 60F 254, glss, lyer thickness 0.25 mm Merck RP-18 F 254s, glss, lyer thickness 0.2 mm Merck RP-2 F 254s, glss, lyer thickness 0.2 mm Prior to chromtogrphic seprtion, RP-18 nd RP-2 pltes were pre-wshed with methnoldichloromethne 1:1 (v/v) nd dried t room temperture for 24 h. Pltes covered with silic gel 60F 254 were used without pretretment. Instrumenttion TLC mnul smpler ñ Cmg, 1 µl cpillries; verticl chromtogrphic chmber; UV lmp 254 nm; densitometer ñ Desg, model CD60. Spotting nd development of chromtogrms UV filters under investigtion were spotted s 0.5 mg/ml solutions in methnol, except for ET

Strtegies of sunscreen seprtion by thin lyer chromtogrphy 793 (cetone), PBS nd PDT (wter + NOH q. 1 mol/l for neutrliztion), 1 µl of the solution per spot. The pltes were spotted 20 mm from the bottom edge nd developed to the distnce of 60 mm from the strt line. Chromtogrms were dried t room temperture, exmined under the UV lmp nd scnned with the densitometer (Ñmultiwvelength scnî mode, wvelength 200ñ400 nm t 20 nm intervls). R f vlues for the prticulr UV filters re listed in Tbles 1ñ4. RESULTS AND DISCUSSION Comprison of R f for 15 UV filters most commonly used in the Europen Community on silic gel 60, RP-18 nd RP-2 sttionry phses led to the conclusion tht seeking the chromtogrphic conditions suitble to seprte ll substnces under investigtion cn be hrdly successful tsk. It ws decided tht from the formultorís point of view it is essentil to propose chromtogrphic conditions suitble for seprtion of such combintions of UV filters tht re prticulrly populr in contemporry cosmetic products: (1) OS + OMC + BZ-3; (2) DHHB + ET + OMC; (3) MBC + OMC + AVO (4) OMC + OCR + AVO; (5) BZ-3 + OMC + OCR; (6) ET + OCR + AVO; (7) PBS + AVO + HMS. Such filter systems re commonly used by cosmetic mnufcturers becuse they offer protection ginst UV- A nd UV-B rys nd reltively high level of the formultion photostbility (12ñ14). On the other hnd, these filter systems, pprently trivil, re, ccording to some reserch, difficult to seprte by HPLC on RP-18 sttionry phse (15, 16). TLC seprtion of some of these combintions in the conditions mentioned erlier (RP-18, methnol-tetrhydrofurne-wter 50:35:15 (v/v/v)) is lso not entirely successful (R f for OCR ñ 0.43 (10); R f for OMC ñ 0.35 (6); R f for AVO ñ 0.37 (17); R f for MBC ñ 0.48 (17)). Comprison of R f vlues for the components of sunscreen combintions (1)ñ(6) mentioned bove obtined under different chromtogrphic conditions suggests tht seprtion of (4) is prticulrly difficult. In other cses the best result were chieved in the following conditions: (1) ñ RP-2, cyclohexne-piperidine 15:1 (v/v) ñ R f (OS) 0.67; R f (OMC) 0.53; R f (BZ-3) 0.42; (2) ñ Silic gel 60, cyclohexne-diethyl ether-i- PrOH 15:1:1 (v/v/v) ñ R f (DHHB) 0.42; R f (ET) 0.23; R f (OMC) 0.55 nd cyclohexne-diethyl ether-cetone 15:1:2 (v/v/v) ñ R f (DHHB) 0.30; R f (ET) 0.15; R f (OMC) 0.47 (18); (3) ñ RP-2, cyclohexne-piperidine 15:1 (v/v) ñ R f (MBC) 0.60; R f (OMC) 0.53; R f (AVO) 0.25; (5) ñ RP-18, methnol-wter 9:1 (v/v) ñ R f (BZ-3) 0.44; R f (OMC) 0.23; R f (OCR) 0.33, cetonitrile- Tble 1. R f vlues for sunscreens on RP-2 sttionry phse. iproh/ MeOH/ Cyhex. MeOH/ MeCN/ n-hexne/ / Cyhex. b / Cyhex. b / Cyhex. b / AcOEt/ THF/ ether H 2 O H 2 O ether / piperidine ether ether H 2 O H 2 O piperidine 9:1 9:1 1:1 15:1 10:1 15:1 6:1:2 50:35:15 15:1:1 OMC 0.77 0.75 0.70 0.88 0.78 0.60 0.53 0.48 0.42 PABA 0.88 ñ ñ ñ ñ 0 0 0 0 OCR 0.78 0.73 0.72 0.89 0.83 0.62 0.57 0.56 0.50 AVO 0.78 0.75 0.72 0.90 0.80 0.30 (*) 0.25 0.56 0.50 MBC 0.78 ñ 0.73 0.88 0.88 0.67 0.60 0.53 0.48 HMS 0.75 0.70 0.70 0.88 0.92 0.70 0.68 0.73 0.70 ET 0.80 0.55 0.42 0.76 0.42 0.15 (*) 0.12 0 0 PBS 0.95 0.98 0.99 0.98 0 0 0 0 0 BZ3 0.78 0.80 0.78 0.94 0.84 0.45 0.42 0.53 0.50 IMC 0.78 0.78 0.77 0.94 0.88 0.57 0.53 0.44 0.38 DHHB 0.78 0.75 0.72 0.88 0.82 0.52 0.50 0.37 0.30 BZ4 0.93 0.97 0.99 0.98 0 0 0 0 0 OS 0.75 0.71 0.69 0.88 0.95 0.70 0.67 0.72 0.70 ODP 0.75 0.72 0.69 0.85 0.86 0.58 0.53 0.44 0.38 PDT 0.87 0.96 0.98 0.88 0 0 0 0 0 diethyl ether, b cyclohexne. All solvents in volumetric rtios.

794 ANNA W. SOBA SKA nd ELØBIETA BRZEZI SKA Tble 2. R f vlues for sunscreens on RP-18 sttionry phse. MeOH/ MeOH/ DMF/ Dioxne/ MeCN/ THF/ Acetone/ MeOH/ MeOH/ MeOH/ EtOH/ MeOH/ MeOH/ EDTA b. ph 3 H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O b. ph 13 b NH 3 2M H 2 O/ MeCN THF/H 2 O MeOH/ iproh/ THF/ AcOEt/ 9:1 9:1 9:1 9:1 9:1 9:1 9:1 (0.002M) 9:1 9:1 AcOH 9:1 50:35:15 b. ph 13 H 2 O 9:1 70:29.5:0.5 50:35:15 6:1:2 OMC 0.40 0.73 0.34 0.79 0.59 0.23 0.21 0.25 0.21 0.22 0.07 0.50 0.37 0.39 0.47 PABA 0.98 0.95 0.97 0.90 0.97 0.89 0.86 0.88 ñ 0.85 ñ ñ 0.86 0.87 ñ OCR 0.50 0.77 0.40 0.83 0.68 0.33 0.32 0.35 0.30 0.34 0.12 0.61 0.42 0.45 0.54 AVO 0.52 0.74 0.32 (*) 0.83 0.65 0.22 (*) 0.21 (*) 0.23 (*) 0.20 (*) 0.22 (*) 0.08 (*) 0.50 (*) 0.37 (*) 0.40 (*) 0.44 MBC 0.56 0.74 0.45 0.83 0.68 0.34 0.34 0.37 0.31 0.37 0.16 0.57 0.48 0.50 0.55 HMS 0.36 0.65 0.30 0.78 0.55 0.20 0.18 0.20 0.18 0.20 0.05 0.47 0.30 0.32 0.40 ET 0.03 0.61 0 0.79 0.34 0 0 0 0 0 0 0.18 0.06 0.06 0.24 PBS 0.98 0.84 0.97 0.97 0.98 0.92 0.92 0.93 0.89 0.90 0.88 0.91 0.98 0.95 0.98 BZ3 0.75 0.82 0.67 0.85 0.76 0.44 0.45 0.47 0.44 0.45 0.30 0.63 0.53 0.56 0.63 IMC 0.60 0.79 0.54 0.84 0.72 0.35 0.36 0.39 0.34 0.37 0.18 0.61 0.48 0.50 0.57 DHHB 0.54 0.79 0.39 0.84 0.70 0.33 0.34 0.39 0.34 0.36 0.17 0.61 0.47 0.48 0.63 BZ4 0.98 0.93 0.86 0.98 0.97 0.90 0.88 0.93 0.86 0.90 0.82 0.94 0.98 0.91 0.98 OS 0.34 0.65 0.33 0.79 0.58 0.19 0.18 0.21 0.19 0.22 0.07 0.50 0.30 0.32 0.42 ODP 0.39 0.70 0.32 0.82 0.63 0.21 0.21 0.23 0.20 0.25 0.06 0.50 0.37 0.40 0.50 PDT 0.98 0.98 0.94 0.90 0.98 0.95 0.97 0.98 0.98 0.98 0.98 0.98 0.98 0.96 0.98 buffer ph 13 b buffer ph 3, (*) ñ pek tiling. All solvents in volumetric rtios.

Strtegies of sunscreen seprtion by thin lyer chromtogrphy 795 Tble 3. R f vlues for sunscreens on silic gel 60 sttionry phse. AcOEt/ AcOEt/ AcOEt/ Cyhex. MeCN/ / Cyhex. / EtOH/ EtOH/ EtOH/ ether H 2 O b / ether b / H 2 O H 2 O H 9:1 2 O/AcOH iproh AcOEt 14:7:6 14:10:3 14:7:5:1 15:1:1 15:1:1 Cyhex. / Cyhex. / Cyhex. / Cyhex. / Cyhex. / ether b ether b ether b ether b piperidine 1:1 5:1 10:1 15:1 15:1 OMC 0.92 0.89 0.94 0.94 0.55 0.36 0.56 0.37 0.29 0.17 0.30 PABA 0.90 0.90 0.68 0.88 0.06 0 0.12 0 0 0 0 OCR 0.91 0.91 0.93 0.93 0.55 0.36 0.60 0.40 0.34 0.15 0.29 AVO 0.90 0.91 0.93 0.93 0.55 0.33 0.58 0.37 0.30 0.20 0.03 MBC 0.88 0.91 0.92 0.93 0.55 0.43 0.60 0.43 0.42 0.22 0.35 HMS 0.91 0.92 0.93 0.93 0.68 0.67 0.88 0.63 0.60 0.53 0.37 (*) ET 0.98 0.97 0.96 0.95 0.23 0.02 0.23 0 0 0 0 PBS 0.58 0.63 0.53 (*) 0.60 0 0 0 0 0 0 0 BZ3 0.89 0.92 0.87 0.93 0.54 0.36 0.57 0.37 0.37 0.23 0.10 IMC 0.91 0.92 0.90 0.93 0.51 0.31 0.60 0.30 0.24 0.13 0.27 DHHB 0.91 0.91 0.92 0.93 0.42 0.20 0.37 0.20 0.11 0 0.22 BZ4 0.60 0.62 0.60 (*) 0.63 0 0 0 0 0 0 0 OS 0.92 0.92 0.92 0.93 0.69 0.67 0.88 0.62 0.60 0.53 0.36 (*) ODP 0.91 0.92 0.92 0.93 0.49 0.31 0.57 0.32 0.25 0.13 0.29 PDT 0.16 0.15 (*) 0.30 (*) 0.09 (*) 0 0 0 0 0 0 0 cyclohexne, b diethyl ether, (*) ñ pek tiling. All solvents in volumetric rtios.

796 ANNA W. SOBA SKA nd ELØBIETA BRZEZI SKA Tble 4. R f vlues for sunscreens on silic gel 60 sttionry phse ñ continued. Cyhex. / Cyhex. / Cyhex. / Cyhex. / Cyhex. / Cyhex. / Toluene/ Toluene/ Toluene/ Cyhex. / Cyhex. / ether b / ether b / cetone cetone cetone 15:1:1 15:1:2 ether b / AcOEt AcOEt/ AcOEt/ ether iproh AcOEt CHCl 3 Toluene /iproh/ ether b / AcOH AcOH AcOEt iproh 15:1 15:1 1:1 15:1 15:1:3 15:1:0.2 15:1:1 15:1:1:1 15:1:0.2 OMC 0.41 0.47 0.52 0.48 0.34 0.37 0.27 0.60 0.60 0.67 0.68 0.45 PABA 0 0 0.03 ñ ñ 0 0 ñ ñ ñ ñ ñ OCR 0.34 0.44 0.43 0.51 0.34 0.38 0.27 0.68 0.67 0.72 0.67 0.41 AVO 0.34 0.42 0.42 0.51 0.31 0.38 0.33 0.50 (*) 0.63 (*) 0.67 (*) 0.62 0.37 MBC 0.48 0.56 0.58 0.50 0.42 0.38 0.22 0.54 0.55 0.63 0.71 0.46 HMS 0.64 0.68 0.68 0.65 0.66 0.66 0.63 0.82 0.82 0.82 0.86 0.77 ET 0.02 0.15 0.21 0.10 0 0 0 0.08 0.12 0.36 0.29 0 PBS 0 0 0 0 0 0 0 0 0 0 0 0 BZ3 0.37 0.41 0.41 0.54 0.35 0.42 0.33 0.56 0.60 0.66 0.62 0.43 IMC 0.33 0.42 0.44 0.50 0.29 0.37 0.22 0.48 0.52 0.60 0.61 0.34 DHHB 0.21 0.30 0.37 0.43 0.17 0.16 0.06 0.32 0.37 0.48 0.51 0 BZ4 0 0 0 0 0 0 0 0 0 0 0 0 OS 0.64 0.66 0.64 0.66 0.65 0.66 0.63 0.80 0.82 0.81 0.88 0 ODP 0.33 0.42 0.46 0.41 0.29 0.32 0.22 0.50 0.50 0.58 0.59 0.34 PDT 0 0 0 0 0 0 0 0 0 0 0 0 cyclohexne, b diethyl ether, (*) ñ pek tiling. All solvents in volumetric rtios..

Strtegies of sunscreen seprtion by thin lyer chromtogrphy 797 wter 9:1 (v/v) ñ R f (BZ-3) 0.67; R f (OMC) 0.34; R f (OCR) 0.40, dimethylformmide-wter 9:1 (v/v) ñ R f (BZ-3) 0.75; R f (OMC) 0.40; R f (OCR) 0.50. The presence of AVO mde the TLC seprtion of (6) prticulrly complicted becuse this UV filter in some conditions (e.g., RP-18 sttionry phse nd methnol-wter 9:1 (v/v) or methnol-cetonitrile 9:1 (v/v) s mobile phse) tends to give somewht streked spots tht overlp with those of OCR, despite the pprent difference in R f vlues. During the investigtion of the influence of different orgnic solvents dded to cyclohexne on the retention of UV filters on silic gel 60, it ws estblished tht the ddition of piperidine to mobile phse modifies the retention properties of sunscreens to vrious degrees. Using mobile phse cyclohexne-piperidine 15:1 (v/v) insted of cyclohexnediethyl ether 15:1 (v/v) leds to the increse in R f for OMC, OCR, MBC, IMC, DHHB nd ODP, wheres the vlues of R f for AVO, HMS, OS nd BZ-3 decrese. On the bsis of this observtion it ws suggested tht the combintion of OCR, AVO nd ET should be nlyzed with seprte chromtogrms for OCR (silic gel 60, mobile phse ñ cyclohexne-piperidine 15:1 v/v) nd AVO + ET (silic gel 60, cyclohexne-ether 1:1 v/v) (19). In this sitution, fter the development of chromtogrm with cyclohexne nd piperidine, AVO remins ner the strt line nd its spots do not interfere with densitometric quntifiction of OCR. It ws estblished tht highly hydrophilic sunscreens (PDT nd PBS) cn be seprted on silic gel 60 with mobile phses: ethyl cette-ethnolwter 14:7:6 (v/v/v) or cetonitrile-wter 9:1 (v/v), wheres the seprtion of PBS nd BZ-4 in these conditions is impossible. When formultion contins both hydrophilic (e.g., PBS, PDT or BZ-4) nd lipophilic (e.g., AVO, HMS) UV filters (see the filter combintion (7)) it is resonble to seprte them by multiple-development TLC on silic gel 60 using the mobile phse of lower polrity first (e.g., diethyl ether-cyclohexne) followed by the phse of higher polrity (e.g., ethyl cette-ethnol-wter). If such procedure is employed, hydrophobic filters migrte first nd the hydrophilic ones cn be nlyzed fter the second development of chromtogrms (20). CONCLUSIONS The conditions proposed in this pper my be employed to seprte nd subsequently quntify UV filters commonly used in UE, especilly in the following combintions: AVO-OCR-ET, PBS-HMS- AVO, DHHB-OMC-ET nd MBC-OCR-AVO. The pplicbility of the proposed conditions ws proved during the nlysis of some cosmetic products vilble in Polnd (18ñ20). Acknowledgments This work ws supported by n internl grnt from the Medicl University of Lodz, Polnd (no. 503/3-016-03/501-01). Thnks re due to Merck, BASF nd Symrise for free smples of sunscreens used in this investigtion. REFERENCES 1. Slvdor A., Chisvert A.: Anl. Chim. Act 537, 1 (2005). 2. Slvdor A., Chisvert A.: in Anlysis of Cosmetic Products, Slvdor A., Chisvert A. Eds., pp. 94ñ121, Elsevier, Amsterdm 2007. 3. Eiden F., Tittel Ch.: Dtsch. Apoth. Ztg. 121, 2693 (1981). 4. Eiden F., Tenczer J.: Dtsch. Apoth. Ztg. 111, 118 (1971). 5. Liem D.H.: J. Soc. Cosmet. Chem. 27, 163 (1976). 6. Musil B., Sherm J.: Act Chromtogr. 8, 4 (1988). 7. Westgte E., Sherm J.: Am. Lb. 13 (2000). 8. Westgte E., Sherm J.: J. Liq. Chromtogr. Rel. Technol. 23, 609 (2000). 9. Musil B., Sherm J.: J. Plnr Chromtogr. 10, 368 (1997). 10. Fisher J, Sherm J.: J. Plnr Chromtogr. 13, 388 (2000). 11. Bumgrtner V., Hohl Ch., Huri U.: J. Plnr Chromtogr. 22, 193 (2009). 12. Mendrok-Edinger Ch., Smith K., Jnssen A., Vollhrdt J.: Cosmet. Toiletr. 124, 47 (2009). 13. Gsper L.R., Mi Cmpos P.M.B.G.: Int. J. Phrm. 307, 123 (2006). 14. Bond C.: Cosmet. Toiletr. 123, 49 (2008). 15. Chisvert A., Pscul-Mrt M.C., Slvdor A.: J. Chromtogr. 921, 207 (2001). 16. Scli S.: J. Phrm. Biomed. Anl. 38, 250 (2005). 17. Polczyk A.: M. Phrm. Thesis, Fculty of Phrmcy, Medicl University of Lodz, 2009. 18. SobÒsk A.W., BrzeziÒsk E.: J. Plnr Chromtogr. in press. 19. SobÒsk A.W., BrzeziÒsk E.: J. Plnr Chromtogr. 24, 154 (2011). 20. Dereck K.: M. Phrm. Thesis, Fculty of Phrmcy, Medicl University of Lodz 2010. Received: 20. 05. 2011