Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors

Accepted Manuscript Title: Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors Author: Patr´ıcia Mazureki Campos Fab´ıola Silva Garcia Prac¸a Maria Vit´oria Lopes Badra Bentley PII: DOI: Reference:

S1570-0232(15)30096-9 http://dx.doi.org/doi:10.1016/j.jchromb.2015.07.029 CHROMB 19529

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

18-2-2015 11-7-2015 13-7-2015

Please cite this article as: Patr´icia Mazureki Campos, Fab´iola Silva Garcia Prac¸a, Maria Vit´oria Lopes Badra Bentley, Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors

Patrícia Mazureki Camposa, Fabíola Silva Garcia Praçab, Maria Vitória Lopes Badra Bentleyc,*

a

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil. E-mail: [email protected]. Phone: +55 16 3315 4302. b

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil. E-mail: [email protected]. Phone: +55 16 3315 4302. c

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil. * Corresponding author. Phone: +55 16 3315-4302. Fax: +55 16 3315-4181. E-mail address: [email protected].

Highlights

We developed extractionmethod for lipoic acid from skin samples. HPLC/ELS first time used for lipoic aciddue to characteristic detection. HPLC/EC is useful to LA assays as in vitro permeation and skin bioavailability studies. HPLC/UV can be used forstability studies and formulation design of lipoic acid. We reportedthe validation of three different HPLC assaysfor lipoic acid.

ABSTRACT Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection.

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These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 x 4 mm, 5 µm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71 %. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 µg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 µg/mL, respectively, for HPLC/EC for the added LA concentration of 6.67 µg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 µg/mL, respectively, for HPLC/ELS for the added LA concentration of 10 µg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

Keywords: Lipoic acid; HPLC-ultraviolet; HPLC-electrochemical: HPLC-evaporative light scattering; Validation; Skin samples.

1. Introduction Alpha-lipoic acid (LA) is a disulphide carboxylic compound [5-(1,2-dithiolan-3yl)pentanoic acid], the oxidation product of dihydrolipoic acid (DHLA) (Scheme 1). LA and DHLA occur naturally in mitochondria, act as a coenzyme for dehydrogenase reactions, and are also aminomethyl carriers in the glycine-cleavage enzyme system [1]. The LA/DHLA system is considered a potent antioxidant, which scavenges reactive oxygen species and modulates the redox state of cells. It is considered to be an ideal therapeutic antioxidant because it exists naturally, has a low molecular weight and is effective in both aqueous and lipid environments [2,3]. LA is readily converted into DHLA inside cells (Scheme 1). The redox potential of this pair is about – 0.32 V; consequently, it is able to reduce the oxidized species, such as glutathione disulfide (GSSG) to glutathione (GSH), another important cell antioxidant system. LA also serves as a coenzyme for the reduction for NADP+/NADPH, which shows how LA is coupled to the cell metabolism and redox state [4,5]. The therapeutic potential of LA/DHLA is related to its antioxidant properties and has been used in the treatment of several diseases, such as diabetes, cancer, cardiovascular, neurodegenerative, and autoimmune diseases [6]. The most challenging aspect of drug application into/through the skin is the low penetration characteristics of some drugs including LA. As a result of this, topical and transdermal delivery systems for LA are required [7,8] to improve the entry of the drug into skin. LA´s lipid-soluble and water-soluble properties favor rapid penetration in all compartments of the cell and tissues [9]. The drug concentration in the skin and its cutaneous bioavailability are fundamental to the performance studies of topical formulations because a considerable drug amount is necessary to produce local effect

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[10]. Although there are reports showing therapeutic applications of LA on the skin [11,12], there is no publication describing its extraction and quantification from the skin without labeling techniques. Indeed, the topical application of antioxidants could be more effective and practical for the treatment of skin pathologies [8]. Taking into account all reasons, the proper quantification of LA is crucial for determining its bioavailability in the skin, as well as during the screening and designing of new topical formulations. The analytical methods already reported for LA include GC, colorimetry, CE and HPLC. The last one is more accessible in practice. Based on the detection method, there are specific characteristics and limitations [1,13]. The following methods have been used for the quantification of LA. (i) Fluorescence detection in a study of supplementation of LA to old and young volunteers [14]. But this method needs a derivatization procedure, which is laborious. (ii) Ultraviolet absorbance detection for the evaluation of the encapsulation efficiency of LA in solid lipid nanoparticles [15], even without a strong chromophore. (iii) Electrochemical detection via the LA/DHLA inter-conversion due to the application of an electric potential to samples of human plasma [16]. Thus far, LA has not been detected using evaporative light scattering (ELS), a method based on the refractive index of non-volatile substances [17]. In addition to the quantification requirements of the low LA concentration in the skin, the lipid composition of this site can cause problems. The objectives of the present study were to optimize, validate and compare three detection methods with HPLC for the determination of LA obtained from skin samples (absorption membrane), as well as the validation of extraction procedures of LA from skin samples, considering small amounts of LA. Further, to supply options with different detection methods according to lab availability.

2. Experimental

2.1. Chemical and reagents Lipoic acid (≥ 99%) was purchased from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile was chromatographic grade and purchased from J. T. Baker (Phillips-burg, NJ, USA). Water was purified on a Milli-Q system (Millipore, Bedford, MA, USA). Acetic acid and sodium dihydrogenphosphate were purchased from Synth (Diadema, Sao Paulo, Brazil).

2.2. Skin sample preparation The skin from the outer surface of a freshly excised porcine ear (local slaughterhouse, Ipuã – SP) was carefully dissected (making sure that the subcutaneous fat was maximally removed), stored at −20 °C and used within a month.

2.3. Apparatus and chromatographic conditions 3

The study was carried out using an HPLC Shimadzu (Kioto, Japan), equipped with an LC-AD pump, autosampler model SIL-10AF, model CTO-10A column oven, and model SCL-10A controller system. Ultraviolet (UV) detector model SPD – 10AVP. Electrochemical (EC) detector model L-ECD-6A (cell volume – 3.7 µL, working electrode – glassy carbon, reference electrode – Ag/AgCl, auxiliary electrode – SUS316). Evaporative light scattering (ELS) detector model ELSD-LT. The HPLC system was controlled by the Class-VP software.

Chromatographic separations were performed using a LiChrospher®100 RP-18 (250 x 4 mm, 5 µm) with pre-column RP-18 (4 x 4 mm, 5 µm; Merck, Darmstadt, Germany) maintained at 40 °C. Isocratic elution at a flow rate of 0.8 mL/min for HPLC/UV and HPLC/EC and 0.6 mL/min for HPLC/ELS was performed. The quantification of LA was achieved using a UV detector operating at 340 nm [18] or an EC detector with amperometric detection at high oxidation potential of +1.1 V [19]. The ELS detector was set to gain of 10, the drift tube temperature to 40 °C, and the internal compressed air pressure to 300 kPa. The HPLC/UV mobile phase was acetonitrile-0.1 M dihydrogenphosphate (50:50, v/v; pH 2.5). The HPLC/EC mobile phase was acetonitrile-dihydrogenphosphate acetonitrile-0.1 M dihydrogenphosphate (60:40, v/v; pH 2.5). The HPLC/ELS mobile phase was acetonitrile-0.1 M acetic acid (60:40,

v/v; pH 2.5). The run time was 8 min, and the injection volume was 20 µL for the UV, EC and ELS detections. In the HPLC/UV and HPLC/ELS methods LA (oxidized form) was detected. In the HPLC/EC method DHLA (reduced form) was detected after application of oxidation potential from the EC system detector.

2.4. Preparation of standard solutions The primary stock solution of LA was daily prepared by dissolving 2 mg of LA in 2 ml acetonitrile. The working solutions of LA were prepared via the appropriate dilution of the stock solution using acetonitrile as described below. All solutions were prepared in amber flasks and protected from light (covered with aluminum foil).

2.5. Comparative validation parameters of LA quantification using HPLC and ultraviolet, electrochemical or evaporative light scattering detectors

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2.5.1. Selectivity The selectivity of the methods was verified in the presence of potentially interfering molecules, such as acetonitrile, the mobile phase, a solution of skin homogenate and adhesives tape. The absence of interfering peaks at the retention time of LA was considered acceptable selectivity.

2.5.2 Linearity Calibration curves were prepared from LA in acetonitrile (1 mg/mL) over a range of 0.9 – 10.0 µg/mL for HPLC/UV, 0.13 – 8.0 µg/mL for HPLC/EC and 4.0 – 100.0 µg/mL for HPLC/ELS. The samples were assayed using the chromatographic conditions described above. Calibrations curves were constructed from at least 5 concentration levels to evaluate the linearity of the method, and the response ratios (peak heights) were plotted against their respective concentrations using a linear least squares regression to generate the standard calibration equation.

2.5.3. Precision and accuracy Precision and accuracy were evaluated through repeated injections at three LA (in acetonitrile) concentrations of: 6, 2, 1 µg/mL for HPLC/UV; 8, 2, 0.5 µg/mL for HPLC/EC; 75, 25, 8 µg/mL for HPLC/ELS on the same day (intra-assay) and on three different days (inter-assay), producing 9 replicates for each LA concentration. The accuracy (recovery, %) was calculated by means of the Formula:

Accuracy = (measured concentration / nominal concentration) x 100

2.5.4. Sensitivity

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The sensitivity was determined based on the lower limits of detection (LLOD) and quantification (LLOQ). The LLOD was the concentration at which the signal-to-noise ratio (S/N) was 3:1. The LLOQ was evaluated based on the quantification of known concentrations of LA and by determining the minimum level at which LA can be quantified with accuracy and precision. They were determined from their respective chromatograms using software.

2.5.5. Extraction studies for lipoic acid from skin The extraction of LA from skin of porcine ears was based on a previously described methodology [20]. To separate the stratum corneum (SC) from the remaining epidermis+dermis (EP+D), skin sections were subjected to tape stripping (15 pieces of adhesive tape - 3M, Scotch Book Tape no. 845; 3M, St Paul, MN, USA). Then, 40 µL of a solution of 1000 µg/mL LA in acetonitrile was applied to the skin samples – SC (n=3) and EP+D (n=3). It was allowed to dry for 2 min under an air flow. Subsequently, skin samples were collected in 6 mL acetonitrile (HPLC/UV and HPLC/EC) or in 4 mL acetonitrile (HPLC/ELS), vortex stirred for 2 min and put into ultrasound water bath for 15 min. The remaining EP+D was cut into small pieces, the volumes of acetonitrile 6 mL for HPLC/UV and HPLC/EC or 4 mL acetonitrile for HPLC/ELS were added. The samples were vortex mixed for 2 min and grinded for 1 min through a Ultra Turrax tissue cutter. Then, they were put into ultrasound water bath for 15 min and centrifuged at 1360 x g for 5 min. Finally, the samples were filtered using a 0.45-µm membrane and the LA was assayed by HPLC.

3.

Results and discussion

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Several chromatographic methods have been reported for the analysis of LA in various biological samples except for skin. Yet, no HPLC/ELS method has been reported for LA thus far. HPLC/ELS is a useful method employed to determine the lipid composition of different samples and the purity of lipid classes [21]. LA assays are essential for the development of drug delivery systems and for supporting the treatment for skin pathologies [22]. This is because knowledge of the exact LA amount present in the skin and its main layers, as the outermost layer, the stratum corneum, and the deeper layer, the dermis, is important for successful therapy. LA methods of analysis allow choosing an adequate topical formulation, the therapeutic dose, as well as to know LA bioavailability in the skin. For these reasons, we developed and validated HPLC methods considering the specific requirements as recommended in standard guidelines [23, 24]. The experimental conditions have been optimized to improve the sensitivity, retention time and resolution of the peaks. We adjusted the proportion of solvents used in the mobile phase, the oven temperature and the flow rates for the three methods (data not shown). The validation parameters for three HPLC methods are shown in Table 1. The results from analyses of the calibration curves are shown in Figure 1.

3.1.

LA analysis by HPLC/EC

Specifically, in the HPLC/EC, the oxidation potential was adjusted to +1.1 V, and the concentration of the sodium dihydrogenphosphate solution was increased to 0.1 M, which gave the highest response for LA. The mobile phase was degassed with helium to remove oxygen, which can interfere with LA

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reduction. The LA peak presented a slight tail due the adsorption of LA on electrode surface. This phenomenon did not impair the method reliability and is in accordance with data reported previously [18]. LA displayed linearity over a wide concentration range. Intra- and inter-day precision (coefficient of variation) was less than 6.5%. The accuracy of this method was greater than 90%, determined from the percentage recovery at 8, 2, 0.5 µg/mL LA. The LLOD and LLOQ values were determined to be 1.0 and 0.91 ± 0.05 µg/mL, respectively. The chromatograms for standard solutions and LLOQ of LA determined by HPLC/EC are shown in Figure 2A. Routine cleaning of the surface of the electrode was performed to maintain the sensitivity [19]. In this way, the results obtained from skin analyses are reproducible and accurate as reported for LA in plasma samples [25]. Therefore, HPLC/EC may be applied to samples with low concentrations of LA.

3.2.

LA analysis by HPLC/ELS

The HPLC/ELS parameters were set and maintained steady using the chromatographic conditions of the isocratic system. Gain, temperature of the drift tube and internal pressure were adjusted until a smooth baseline was obtained. Critical situations were observed by varying the internal pressure of the compressed air and the optimal value was reached at 300 ± 5.0 kPa. The drift tube temperature was set to 40 °C to evaporate the mobile phase. All conditions were kept the same for all analyses. HPLC/ELS is widely used and its principle is well described [26]. Nonlinearity is a feature of the ELS detector; thus, non-linear curves have been

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described for several compounds when analyzed using ELS detectors [27]. Nevertheless, the lack of linearity did not exclude the use of ELS detection for LA quantification in skin, considering that the non-linear curves include a linear interval. Another characteristic is that the detector response depends on the mass of the analyte; therefore, it is necessary to prepare individual curves for each substance [28]. The linear interval for HPLC/ELS was observed at the concentrations tested, which were included into calibration curve (4 – 100 µg/mL) for this method (Fig. 3). We chose to adjust the linearity via transformation to a logarithmic peak height scale, as well logarithmic transformation of LA concentrations (Fig. 1C). In addition, the expression of detector response is mass on column, which is calculated in function of LA solution concentration and injected volume [28]. Therefore, the concentrations of the LA standard solutions were 100, 75, 50, 25, 10, 8, 6, 4 µg/mL, which correspond to 2, 1.5, 1, 0.5, 0.2, 0.16, 0.12, 0.08 ng on column (injected volume 20 µL). Further, it was possible to obtain a linear correlation that excludes the non-linear character of the detector and enables quantitative analysis. The precision was satisfactory (less than 6.6 and 9.5 %) for intra- and inter-day measurements. The accuracy values were close to 100% for the three assayed LA concentrations. Furthermore, the superiority of HPLC/ELS method lies on the sensitivity in terms of LLOQ/LLOD by mass on the column, which is on the order of nanograms because 4 µg/mL LA is equivalent to 0.08 ng on the column (calculated based on the injection volume of the sample). The chromatograms of standard solutions and LLOQ for LA evaluated for HPLC/ELS are presented in Figure 2B. In fact, this method was developed to assess lipid, non-volatile compounds [29], and offered rapid and sensitive chromatographic separations.

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Because skin is rich in lipids, it is advantageous to have an option for assessing efficiently LA samples that come from skin.

3.3.

LA analysis by HPLC/UV

For HPLC/UV there was a good precision over the tested LA concentration range. Thus, intra- and inter-day precision ranged between 3% and 8%, respectively. The accuracy was satisfactory. The sensitivity was poor due to the absence of a chromophore. Figure 2C shows chromatograms for standard solutions of LA performed by HPLC/UV. To increase the sensitivity, derivatization has been used, which however requires several steps to obtain fluorescence [30]. The HPLC/UV method is a rapid technique and may be used for LA analyses during formulation design.

3.4.

LA analysis is skin samples

All three HPLC methods were able to quantify precisely and accurately LA from skin, indicating that potentially interfering substances did not impair these assays. Typical chromatograms obtained by using the HPLC methods reported in this work as shown in Figure 4 and Figure 5. These three quantification methods for LA were rapid and easy and practical. The amounts of LA extracted from the skin samples are summarized in Table 2. LA was extracted from the skin with recovery rates of 70% and above. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 µg/mL, respectively for HPLC/UV and 6.52 ± 0.49

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and 5.01 ± 0.41 µg/mL, respectively for HPLC/EC for the added LA concentration of 6.67 µg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 µg/mL, respectively for HPLC/ELS for the added LA concentration of 10 µg/mL). The HPLC/EC method is useful for the quantification of low concentrations of LA in skin samples for in vitro and in vivo studies. HPLC/UV can be used for stability studies of LA at middle concentrations. HPLC/UV has been applied to stability studies on LA encapsulated in polymeric nanoparticles [31]. HPLC/ELS, due to its nature of detection, may be employed for the development of topical formulations of LA at high concentrations. The present work shows that HPLC/ELS is a useful quantification approach for LA skin samples.

4.

Conclusion Three HPLC methods with different detection techniques were optimized

and validated for the analysis of LA from skin samples. All the methods provide good linearity, precision, accuracy, selectivity. HPLC with UV, EC and ELS detection should be useful in vitro and in vivo studies including the development of drug delivery systems for pharmaceutical preparations of LA for use in skin disorders.

ACKNOWLEDGMENTS

This study was supported by a grant from the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq, Brazil). P.M. Campos is a 11

PhD student and would like to thank the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES, Brazil) for fellowship support.

REFERENCES [1] H. Kataoka, Chromatografic analysis of lipoic acid and related compounds, J. Chromatogr. B. 717 (1998) 247 – 262. [2] M. Goralska, R. Dackor, B. Holley, M. C. McGahan, Alpha lipoic acid changes iron uptake and storage in lens epithelial cells, Exp. Eye Res. 76 (2003) 241 – 248. [3] K. Tsuji-Naito, S. Ishikura, M. Akagawa, H. Saeki, α-Lipoic acid induces collagen biosynthesis involving prolyl hydroxylase expression via activation of TGF-β-Smad signaling in human dermal fibroblasts, Connective Tissue Res. 51 (2010) 378 – 387. [4] J. Teichert, R. Preiss, Determination of lipoic acid in human plasma by highperformance liquid chromatography with electrochemical detection, J. Chromatogr. B 672 (1995) 277 – 281. [5] A. I. Haj-Yehia, P. Assaf, T. Nassar, J. Katzhendler, Determination of lipoic acid and dihydroplipoic acid in human plasma and urine by high-performance liquid chromatography with fluorimetric detection, J. Chromatogr. A 870 (2000) 381 – 388. [6] A. Goraca, H. Huk-Kolega, A. Piechota, P. Kleniewska, E. Ciejka, B. Skibska, Lipoic acid – biological activity and therapeutic potential, Pharmacol. Rep. 63 (2011) 849 – 858. [7] M. Podda, H. J. Tritschler, H. Ulrich, L. Packer, α-Lipoic acid supplementation prevents symptoms of vitamin E deficiency, Biochem. Biomed. Res. Commun. 204 (1994) 98 – 104. [8] M. Podda, M. Rallis, M. G. Traber, L. Packer, H. I. Maibach, Kinetic study of cutaneous and subcutaneous distribution following topical application of [7,8-14C]rac-αlipoic acid onto hairless mice. Biochem. Pharmacol. 52 (1996) 627-633. [9] A. Segall, M. Sosa, A. Alami, C. Enero, F. Hormaechea, M. T. Pizzorno, C. Bregni, R. Serrao, Stability study of lipoic acid in the presence of vitamins A and E in o/w emulsions for cosmetic application, J. Cosmet. Sci. 55 (2004) 449 – 461. [10] C. Herkenne, I. Alberti, A. Naik, Y. N. Kalia, F. X. Mathy, V. Préat, R. H. Guy, In vivo methods for the assessment of topical drug bioavailability, Pharm. Res. 25 (2008) 87 – 103. [11] Y. S. Ho, C.S. Lai, H. I. Liu, S. Y. Ho, C. Tai, M. H. Pan, Y. J. Wang, Dihydrolipoic acid inhibits skin tumor promotion through anti-inflammation and antioxidation, Biochem. Pharmacol. 73 (2007) 1786 – 1795.

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[12] J. H. Kim, G. S. Sim, J. T. Bae, J. Y. Oh, G. S. Lee, D. H. Lee, B. C. Lee, H. B. Pyo, Synthesis and anti-melanogenic effects of lipoic acid–polyethylene glycol ester, J. Pharm. Pharmacol. 60 (2008) 863 – 870. [13] T. Inoue, M. Sudo, H. Yoshida, K. Todoroki, H. Nohta, M. Yamaguchi, Liquid chromatographic determination of polythiols based on pre-column excimer fluorescence derivatization and its application to α-lipoic acid analysis, J. Chromatogr. A 1216 (2009) 7564-7569. [14] D. J. Keith, J. A. Butler, B. Bemer, B. Dixon, S. Johnson, M. Garrard, D. L. Sudakin, J. M. Christesen, C. Pereira, T. M. Hagen, Age and gender bioavailability of R- and R,S-α-lipoic acid: a pilot study. Pharmacol. Res. 66 (2012) 199-206. [15] K. C. Kang, N. J. Jeong, C. I. Lee, H. B. Pyo, Preparation and characterization of SLNs (W/O/W type) contained lipoic acid PEG ester by variation lipid, J. Ind. Eng. Chem. 15 (2009) 529-536. [16] A. Khan, M. Khan, Z. Iqbal, L. Ahmad, Y. Shah, D. G. Watson, Determination of lipoic acid in human plasma by HPLC-ECD using liquid-liquid and solid-phase extraction: Method development, validation and optimization of experimental parameters, J. Chromatogr. B 878 (2010) 2782 – 2788. [17] V. L. Cebolla, L. Membrado, J. Vela, A. C. Ferrando, Evaporative light-scattering detection in the quantitative analysis of semivolatile polyciclic aromatic compounds by high-performance liquid chromatography. J. Chromatogr. Sci. 35 (1997) 141-150. [18] S. C. Howard, D. B. McCormick, High-performance liquid chromatography of lipoic acid and analogues, J. Chromatogr. 208 (1981) 129-131. [19] J. Teichert, R. Preiss, High-performance liquid chromatographic assay for αlipoic acid and five of its metabolites in human plasma and urine, J. Chromatogr. B 769 (2002) 269-281. [20] F.S.G. Praça, M. V. L. B. Bentley, M. G. Lara, M. B. R. Pierre, Celecoxib determination in different layers of skin by a newly developed and validated HPLC-UV method, Biomed. Chromatogr. 25 (2011) 1237-1244. [21] L. Chamorro, A. García-Cano, R. Busto, J. Martínez-González, A. Albillos, M. A. Lasunción, O. Pastor, Quantitative profile of lipid classes in blood by normal phase chromatography with evaporative light scattering detector: application in the detection of lipid class abnormalities in liver cirrhosis, Clin. Chim. Acta 421 (2013) 132 - 139. [22] M. Schäfer-Korting, W. Mehnert, H. C. Korting, Lipid nanoparticles for improved topical application of drugs for skin diseases, Adv. Drug Deliv. Rev. 59 (2007) 427– 443. [23] ICH. International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH harmonized tripartite guideline). Validation of analytical procedures: methodology, November 1996. [24] R. Causon, Validation of chromatographic methods in biomedical analysis. Viewpoint and discussion. J. Chromatogr. B 689 (1997) 175-180. 13

[25] A. Khan, Z. Iqbal, D. G. Watson, A. Khan, I. Khan, N. Muhammad, S. Muhammad, H. A. Nasib, N. Iqbal, F. U. Rehman, M. Kashif, Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection. J. Chromatogr. B 879 (2011) 1725-1731. [26] A. Stolywho, H. Colin, M. Martin, G. Guiochon, Study of the qualitative and quantitative properties of the light-scattering detector, J. Chromatogr. 288 (1984) 253275. [27] B. Trathnigg, M. Kollroser, Liquid chromatography of polyethers using universal detectors V. Quantitative aspects in the analysis of low-molecular-mass poly(ethylene glycol)s and their derivatives by reversed-phase high-performance liquid chromatography with an evaporative light scattering detector, J. Chromatogr. A 768 (1997) 223-238. [28] K. Mojsiewicz-Pienkowska, On the issue of characteristic evaporative light scattering detector response. Crit. Rev. Anal. Chem. 39 (2009) 89-94. [29] W. W. Christie, Rapid separation and quantification of lipid classes by high performance liquid chromatography and mass (light-scattering) detection, J. Lipid Res. 26 (1985) 507-512. [30] A. Bernkop-Schnürch, E. Reich-Rohrwig, M. Marschütz, H. Schuhbauer, M. Kratzel, Development of a sustained release dosage form for α-lipoic acid. II. Evaluation in human volunteers, Drug Dev. Ind. Pharm. 30 (2004) 35-42. [31] I. C. Külkamp, K. Paese, S. S. Guterres, A. R. Pohlmann, Estabilização do ácido lipóico via encapsulação em nanocápsulas poliméricas planejadas para aplicação cutânea. Química Nova 32 (2009) 2078-2084.

Figure Legends Scheme 1 Chemical structures of lipoic acid (LA) and dihydrolipoic acid (DHLA) and the potential needed for the inter-conversion.

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Figure 1 Calibration curves for LA using HPLC/UV (A), HPLC/EC (B) and HPLC/ELS (C).

Figure 2 Representation of chromatograms for standard solutions of LA analyzed by: A – HPLC/EC: (1) 8.0 µg/mL, (2) 2.0 µg/mL, (3) 0.5 µg/mL, (4) 0.13 µg/mL – LLOQ; B – HPLC/ELS: (1) 75 µg/mL, (2) 25 µg/mL, (3) 8 µg/mL, (4) 4 µg/mL – LLOQ; C – HPLC/UV: (1) 6.0 µg/mL, (2) 2 µg/mL, (3) 1 µg/mL, (4) 0.9 µg/mL – LLOQ.

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Figure 3 Linear interval of response for LA analysis by HPLC/ELS.

Figure 4 HPLC chromatograms demonstrating the selectivity of LA analysis: A – HPLC/UV: (1) 6 µg/mL LA, (2) mobile phase, (3) epidermis+dermis homogenate, (4) stratum corneum; B – HPLC/EC: (1) stratum corneum, (2)

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epidermis+dermis homogenate, (3) mobile phase, (4) 6 µg/mL LA; C – HPLC/ELS: (1) stratum corneum, (2) epidermis+dermis homogenate, (3) mobile phase, (4) 25 µg/mL LA.

Figure 5 HPLC chromatograms for LA extracted from skin and standard LA: A – HPLC/UV: (1) LA from epidermis+dermis, (2) LA from stratum corneum, (3) 6 µg/mL LA solution; B – HPLC/EC: (1) 6 µg/mL standard LA, (2) LA from epidermis+dermis, (3) LA from stratum corneum; C – HPLC/ELS: (1) LA from epidermis+dermis, (2) LA from stratum corneum, (3) 25 µg/mL standard LA.

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Table 1 Summary of validation parameters for LA in the three HPLC methods. Validation parameters

Detection methods HPLC/UV

HPLC/EC

HPLC/ELS

Linearity Concentration range (µg/mL) Regression equation Correlation coefficient

0.9 – 8

0.13 – 8

4 – 100

y = 74x + 4.9

y = 71495x – 4055.5

y = 1.2x + 3.4

r = 0.9993

r = 0.9987

r = 0.9975

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Precision 6-2-1

8-2-0.5

75-25-8

Inter-day repeatability (n=9)

5.8 ± 0.5; 8.05

7.5 ± 0.2; 2.38

79.8 ± 4,4; 5.54

(LA mean ±SD; CV, %)

1.9 ± 0.1; 7.37

2.3 ± 0.1; 4.37

23.2 ± 2.2; 9.49

0.9 ± 0.02; 2.04

0.5 ± 0.03; 6.54

8.2 ± 0.7; 9.04

Concentration levels (µg/mL)

Intra-day repeatability (n=3)

6.0 ± 0.02; 0.39

8.1 ± 0.13; 1.66

79.2 ± 5.20; 6.57

(LA mean ±SD; CV, %)

1.9 ± 0.06; 3.05

2.0 ± 0.01; 0.53

21.9 ± 0.99; 4.55

1.0 ± 0.01; 1.22

0.5 ± 0.03; 6.64

7.3 ± 0.05; 0.70

Accuracy

103 ± 5 (6 µg/mL)

94 ± 2 (8 µg/mL)

106 ± 4 (75 µg/mL)

(% recovery ± SD; n=9)

107 ± 8 (2 µg/mL)

113 ± 5 (2 µg/mL)

91 ± 8 (25 µg/mL)

107 ± 14 (1 µg/mL)

105 ± 7 (0.5 µg/mL)

99 ± 10 (8 µg/mL)

1.0

1.1

1.0

Sensitivity LLOD (µg/mL) LLOQ (µg/mL) 0.91 ± 0.05 (n=9)

0.13 ± 0.04 (n=9)

4.03 ± 0.20 (n=9)

Table 2 Lipoic acid recovered from skin in the extraction studies Extraction (amount recovered, µg/mL ±

Detection methods HPLC/UV

HPLC/EC

HPLC/ELS

SD; % recovery ± SD; n=3)

19

Nominal LA concentration Stratum corneum Epidermis + dermis

6.67 µg/mL

6.67 µg/mL

10.0 µg/mL

5.41 ± 0.56

6.52 ± 0.49

8.88 ± 0.46

(81.3 ± 8.4)

(97.7 ±7.4)

(88.8 ± 4.6)

4.92 ± 0.33

5.01 ± 0.42

8.95 ± 0.08

(73.8 ± 5.0)

(75.2 ± 6.3)

(89.5 ± 0.8)

20