application of hplc-elsd for the quantification of 5-aminolevulinic acid after penetration into...

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Application of HPLC-ELSD for theQuantification of 5-Aminolevulinic Acidafter Penetration into Human Skin ExVivoVilma Armoškaitė a , Valdas Jakštas b , Liudas Ivanauskas c ,

Almantas Ražukas d , Kristina Ramanauskienė a & Vitalis Briedis a

a Department of Clinical Pharmacy , Lithuanian University of HealthSciences – Medicine Academy , Kaunas , Lithuaniab Department of Pharmacognosy , Lithuanian University of HealthSciences – Medicine Academy , Kaunas , Lithuaniac Department of Analytic and Toxicological Chemistry , LithuanianUniversity of Health Sciences – Medicine Academy , Kaunas ,Lithuaniad Voke Branch, Lithuanian Institute for Agriculture and Forestry ,LithuaniaAccepted author version posted online: 31 Oct 2012.Publishedonline: 01 Mar 2013.

To cite this article: Vilma Armoškaitė , Valdas Jakštas , Liudas Ivanauskas , Almantas Ražukas ,Kristina Ramanauskienė & Vitalis Briedis (2013) Application of HPLC-ELSD for the Quantification of5-Aminolevulinic Acid after Penetration into Human Skin Ex Vivo , Analytical Letters, 46:5, 717-733,DOI: 10.1080/00032719.2012.733898

To link to this article: http://dx.doi.org/10.1080/00032719.2012.733898

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Liquid Chromatography

APPLICATION OF HPLC-ELSD FOR THEQUANTIFICATION OF 5-AMINOLEVULINIC ACIDAFTER PENETRATION INTO HUMAN SKIN EX VIVO

Vilma Armo�sskaite,1 Valdas Jak�sstas,2 Liudas Ivanauskas,3

Almantas Razukas,4 Kristina Ramanauskiene,1 andVitalis Briedis11Department of Clinical Pharmacy, Lithuanian University of Health Sciences– Medicine Academy, Kaunas, Lithuania2Department of Pharmacognosy, Lithuanian University of Health Sciences –Medicine Academy, Kaunas, Lithuania3Department of Analytic and Toxicological Chemistry, Lithuanian Universityof Health Sciences – Medicine Academy, Kaunas, Lithuania4Voke Branch, Lithuanian Institute for Agriculture and Forestry, Lithuania

5-Aminolevulinic acid (5-ALA) has been used for treatment of different skin diseases (e.g.,

skin cancer, actinic keratosis (AK), psoriasis, and acne). The quality of the treatment is

directly associated to the amount of 5-ALA that penetrates into skin. 5-ALA was extracted

from skin samples after performing the experiment with Bronaugh cells for 4 hours. Two

methods were developed by applying different column systems and mobile phase composi-

tions: YMC-Pack Hydrosphere C18 column (250� 4mm, 5lm) in series with Hichrom

Hypersil H5ODS (150� 4mm, 5lm), mobile phase consisted of 0.1% TFA in water with

2% of ACN (method A); ACE 3AQ column (150� 4mm, 3lm) eluted with 0.05% TFA

in water (method B). Exploratory fluorimetric analysis requiring pre-column or post-column

derivatization did not warrant the expectations of simple and effective analysis; therefore,

ELS detection for 5-ALA was considered. Parallel chromatography was applied for

double-validation of the methods in order to correctly evaluate the possibility of ELSD appli-

cation for 5-ALA detection and the quantification amount in skin extracts. The retention

time and adequate lowest limit of quantification (LLOQ) of 5-ALA were determined in both

methods: 5.5min and 20lg/ml (method A); 6.2min and 8.4lg/ml (method B). Both

HPLC-ELSD methods proved to be simple, accurate, precise, reliable, and showed repro-

ducible values in the concentration range of 20–200lg/ml in human skin extracts.

Keywords: 5-aminolevulinic acid; HPLC-ELSD; Parallel validation; Penetration into human skin

Received 3 August 2012; accepted 17 September 2012.

The authors are grateful to Prof. Dr. R. Rimdeika from the Department of Plastic and Reconstruc-

tive Surgery, Hospital of LUHS, Lithuania for supplying the human skin for this research.

Address correspondence to Vilma Armo�sskaite, Department of Clinical Pharmacy, A. Mickeviciaus

9, 3000, Kaunas. E-mail: [email protected]. Present address: School of Pharmaceutical Sciences,

University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneve 4, Switzerland. Email: Vilma.

[email protected]

Analytical Letters, 46: 717–733, 2013

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.733898

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INTRODUCTION

Recognized as one of the most prospectively used prodrugs in photodynamictherapy, 5-aminolevulinic acid (5-ALA) has been used for the treatment of differentdiseases, including skin cancer, psoriasis, acne, various types of neoplasms, super-ficial basal cell carcinoma, squamous carcinoma, Bowen’s disease, actinic keratosis,verrucous hyperplasia, oral leukoplakia, among others (Wiegell and Wulf 2006;Chen et al. 2007; Gold 2008; Uekusa et al. 2010). Various cutaneous pharmaceuticalforms, including solutions, patches, liposomes, microemulsions, various types ofgels, creams, and so forth have been applied (Valenta, Auner, and Loibl 2005;Donnelly et al. 2006; Fang et al. 2010; Manifold and Anderson 2011; Szeimieset al. 2011; Yeung et al. 2011). Different penetration enhancing methods, such asYAG (Yttrium–Aluminum–Garnet) laser, electroporation, iontophoresis, ablation,have been used as well (Lee et al. 2011; L. W. Zhang, Fang, and Fang 2011). In com-parison to solution or lipophilic cream (that possess similar penetration fluxes) gelincreased penetration 2 times; microemulsions, patches 2–4 times; and liposomes,7.5–8 times. Electroporation itself increases the penetration of 5-ALA 2 times, ion-tophoresis and microdermabrasion increases the penetration 5–15 times, and laser(while applying the gel formulation) increases the penetration rate 5—300 times(Armoskaite, Ramanauskiene, and Briedis 2011). Different enhancing techniquesprovide various results; the choice of the enhancing technique or pharmaceuticalform depends on the severity and distributions of AK formations on the skin surface.It is essential to monitor the levels of 5-ALA penetrated into skin while applying dif-ferent transdermal pharmaceutical forms for curing problematic diseases, Quantityof penetrated 5-ALA is an important parameter of optimizing individual treatmentof the diseases previously mentioned.

Several methods for quantifying 5-ALA in solutions, plasma, body fluids, andskin extracts, such as high performance liquid chromatography (HPLC) with fluor-escent detection (Ogasawara et al. 2003; Araujo, Thomazine, and Lopez 2010),MS=MS detection (Zhang 2011), as well as capillary electrophoresis (Bunke et al.2000), have been developed. However, the method of capillary electrophoresis wasnot validated until the end as it was appointed for the determination of 5-ALA degra-dation products. On the other hand, in further studies CE-MS=MS has been success-fully applied for the quantification of 5-ALA and porphobilinogen (Lord, Luo, andLim 2012). HPLCwith fluorescent detection always includes derivatization procedureusing different derivatization agents, for example, acetylacetone and formaldehyde(De Rosa et al. 2003), ortho-phthalaldehyde (OPA) (Winkler and Muller-Goymann2005), or fluorescamine (Namjoshi et al. 2007). The complexity of derivatizationprocedures and production of additional deviations were the main reasons for estab-lishing new, selective and sensitive HPLC assay method solutions, without derivatiza-tion. The powerful MS detection also has following disadvantages: the problematicionization of LC eluate, the carryover and the necessity to use internal standardsor isotopically labeled internal standards. MS detection which requires additionaltechnical solutions is generally applied for samples of low concentration range, there-fore, ELS detection, that requires less specific adjustments, has been selected for themoderate real samples. Hitherto, there has not been any data published aboutapplication of HPLC-ELSD in pharmacy for the development of medicinal product

718 V. ARMOSKAITE ET AL.

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formula regarding the optimization of bioavailability of the active pharmaceuticalingredient that is structurally similar to the components of the target tissue (skin).Therefore, we decided to apply this perspective technique for the quantification of5-ALA in human skin samples.

The reasoning of some parameters of the newly developed method (e.g., speci-ficity) is better secured while comparing the results of different systems that have beenevaluated in parallel. The suitability of applied system components (such as detectiontype) for the analyte is better reasoned if the method conditions differ, therefore, itwas decided to develop two parallel methods that would separately cover two basicprinciples for increasing the theoretical performance for reverse-phase chromato-graphy: by increasing the amount of stationary phase and by decreasing the size ofthe column silica particles.

MATERIALS AND METHODS

Chemicals and Reagents

Pure 5-aminolevulinic acid (purity 98%, lot 039K4119, certificate no. A3785),HPLC grade acetonitrile (ACN), and trifluoroacetic acid (TFA) (purity �98%) havebeen purchased from Sigma-Aldrich (Germany). Sodium chloride 9 g=1000mlsolution for infusion for skin experiments was obtained from Fresenius Kabi(Germany). Carbopol 980 for gel preparations was purchased from Lubrizol Corpor-ation, USA. Base cream (composition: carbomer, triethanolamine, mineral oil, stearicacid, glyceryl stearate) was obtained from The Pharmacy of LUHS (LithuanianUniversity of Health Sciences).

Standard Solutions and Quality Control Samples

Standard solutions for creating calibration curves and validation have beenprepared from pure 5-ALA. To prepare calibration sample, 10mg of the substancewas added to 10ml of purified water. Concentrations for calibration points from20 mg=ml to 150 mg=ml have been prepared by diluting the solution that has been pre-pared before. It was decided to perform calibration every analytical set. Quality con-trol samples were prepared with drug free skin extract at 30 mg=ml (low), 100 mg=ml(medium), and 170 mg=ml (high). All solutions were analyzed in 72 hours (as stabilitytest did not indicate statistically relevant changes) starting from the moment of phar-maceutical base containing 5-ALA application on the skin.

Sample Preparation

Processing the skin. The skin of a Caucasian woman’s (age 45) abdominalarea was obtained after cosmetic surgery in the Department of Plastic and Recon-structive Surgery, Hospital of Lithuanian University of Health Sciences. The studiesthat include human skin were approved by Kaunas Region Bioethical Committee.The skin after removal from the donor was immediately wrapped in aluminum foiland stored at �20�C temperature for not longer than 6 months before use (Katohet al. 2010). The conditions were appropriate for the skin to sustain its main qualities

HPLC-ELSD METHOD FOR THE QUANTIFICATION OF 5-ALA 719

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and for the experiments to be performed with it. Before placing the skin into cells itwas unfrozen in room temperature, rinsed in saline, and hair and fat layer wasremoved with scissors.

Insertion of the skin into equipment. Bronaugh-type flow-through dif-fusion cells have been used for simulating the blood flow in the skin and performingthe experiment correctly (Clowes, Scott, and Heylings 1994). The cells of 0.13mlreceptor volume were mounted with full-thickness human skin. The diffusion areaof single cell was 0.64 cm2. The cells with uploaded pharmaceutical forms (blanksamples) or without them (placebo samples) were placed on the heating block to imi-tate human body temperature (37�C). Equilibration was performed for 12 hours withsaline to obtain the environment closest to human body. After the equilibration per-iod, 200mg of the donor phase, including blank gels and gels with 5-ALA (infinitedose, which equals to 20% w=w) have been applied on the SC (stratum corneum) sideof the skin surface for 4 h. Saline was chosen as an acceptor fluid and it was pumpedat a rate of 0.6ml=min. with the peristaltic pump for 4 h. After this procedure, thedonor phase (blank gel or gel with 5-ALA) was removed and the skin surfacewas rinsed with 2ml of saline. The outer residuals of skin samples were trimmedoff, leaving the central circles with an area of 0.64 cm2.

Separation of skin layers. Dry heat separation method (Kezutyte et al.2010) was used for separating epidermis from the rest of the skin (dermis). The skinsample was placed on the epidermis side on the hot cane (heated up to 60�C) for 2 s.After the both skin layers have been unbounded, the epidermis was peeled off.

Sample extraction. Epidermis and dermis were placed into separate micro-centrifugal tubes and 2ml of purified water with 0.1% TFA was poured into themfor extraction. The samples were left to store in refrigerator for 12 hours. The sampleshave been filtered through 0.2 mm pore filter (diam. 25mm, Nylon SF, Sigma –Aldrich, Germany) before injection to HPLC system. Extraction was optimized byapplying shaking (for 15, 30, 45, 60min), ultrasound bath (for 10, 20, 30, 45min),and comparing these results with adequate extraction without using any of theenhancing methods for the same period of time. Therefore, the extraction procedurewithout application of ultrasound or shaking has been selected. 3 repetitions of thisexperiment have shown none statistically relevant differences. Duration of the extrac-tion was determined by applying 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 14 h. Starting fromthe extraction time of 12 hours, the concentration of 5-ALA which penetrated intoskin from test samples did not increase or decrease statistically relevantly; therefore,12 hours of extraction without application of ultrasound or shaking has been selectedas optimal one.

LC-ELSD system. Chromatographic analysis was performed using a 2695module LC system (Waters, USA), consisting of 2 pumps in series, autosamplerand a degasser. The analyte was detected by ELS detector Alltech 3300 (Alltech,USA). Additionally, the possible impurities of peaks were observed by introducing996 PDA detector (Waters, USA). For the selection of HPLC detection modes fluori-metric detection was applied (474 Scanning Fluorescence Detector, Waters, USA) inprimary steps of method development. Finally, two kinds of columns and adequatemobile phase compositions were introduced and optimized for real samples. For


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the first method (method A) YMC-Pack Hydrosphere C18 column (250� 4mm,5 mm, YMC Co. Ltd., Japan) in series with Hichrom Hypersil H5ODS (150� 4mm,mm, 5 mm, Hichrom, UK) was used. The mobile phase for the first method consistedof 0.1% TFA in water with 2% of ACN. Optimized low temperature ELSD oper-ational characteristics (45�C evaporation temperature, nitrogen flow rate 1.5 l=minand gain of 16) were applied. The second method (method B) was developed by usingACE 3AQ column (150� 4mm, 3 mm, ACT, UK) eluted with 0.05% TFA in water;ELSD evaporation temperature was set on 45�C, nitrogen flow rate and gainwere adequate at 1.5 l=min and 16, respectively. The injection volume of 10 mm wasapplied.

METHOD VALIDATION

Specificity and Identification

For the confirmation of specificity, real samples (extracts from skin after appli-cation of medication), standard solutions, placebo (extracts from skin without anybase and 5-ALA), and blank samples (base solutions) were injected into the HPLCsystem. Characteristics of peak shape, retention time, PDA spectra, matrix effect,and possible overlay of nonspecified impurities have been checked with both meth-ods. Placebo sample consisted of human skin extract without applied gel matrixand without 5-ALA. Blank samples consisted of carbopol base that has been dis-solved in 0.1% TFA solution. Additional separations of active substance (5-ALA)added to the placebo solution obtained from skin extract have been performed.The experiment of 72-hour stability of analytical samples has been performed andmet the data presented in literature (Gadmar et al. 2002); therefore, the degradationproducts of 5-ALA have not been specified in our investigation.

Calibration Curves, Linearity, and Lowest Limit ofQuantification (LLOQ)

According to the literature data (Douville et al. 2006; Peng et al. 2006) moresuitable calibration equations for the dependence of the response of signal (peak area)on concentration of the analyte in ELS detection system are log-log linear and quad-ratic in comparison to the linear ones. The calibration has been integrated by usingprocessing results from at least 5 calibration levels: 20 mg=ml, 40 mg=ml, 50 mg=ml,100 mg=ml, and 200 mg=ml. The optimal curve was selected under the values of R2

(determination coefficient). Additionally, RSS and RSD (E) have been calculatedwith software Empower 2 and facilitated the selection of the suitable equation. Pear-son’s correlation coefficient has been applied as well to approve the relation betweenthe signal area and concentration of 5-ALA in the sample. The data analysis has beenperformed with SPSS 17.0 and Empower 2.

The evaluation of lowest limit of quantification (LLOQ) was based on Signal-to-Noise Approach. The concentration of 30 mg=ml was chosen as a suitable onefor the determination of LLOQ for both methods. The analysis was performed in 3separate days; the average of the sample was calculated. The full application rangeof methods was evaluated by introducing upper limit of quantification (ULOQ).


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ULOQ was determined by transmission and registration of analogical ELS detectorsignal (max. signal registration limit is 2250mV).

Accuracy and Recovery

Three levels of reference solutions were used for analyte recovery evaluation.Standard solution concentration levels cover almost the whole quantification area;the concentrations of these solutions were as follows: range [(30 mg=ml (low),100 mg=ml (medium), and 170 mg=ml (high)] with repetition of 6 times per point intwo sample sets. The acceptance criteria of recovery must be in the diapason of15% (for bioanalytical preparations) (Blume et al. 2011).

Precision

To evaluate the precision of both methods, study samples at three concentrationlevels (30, 100, and 170 mg=ml) were analyzed within run (six replicates) and intra-dayexperiments. The precision was expressed as the relative standard deviation (RSD);the acceptance values used for validation of RSD were within 15%. The acceptancecriterion has been determined by guidelines for bioanalytical methods (EMEA 2010).

RESULTS AND DISCUSSION

Method Development

The concentrations of 5-ALA which appear in skin samples after penetrationand extraction depend on many factors: concentration of 5-ALA that has beenincluded into the formulation, the amount of the formulation, that is put onto theskin, the area of the skin affected by the formulation, the duration of penetrationexperiment, the formulation itself, the additional measures for increasing the pen-etration of 5-ALA (e.g., iontophoresis, laser, penetration enhancers), extraction con-ditions, and so forth (Morrow et al. 2010). The penetration rates of 5-ALA have beenreported to be in a wide diapason, starting from 0.05–0.01 mg=(cm2� h) (Namjoshiet al. 2007) and ending with the interval of 400–450 mg=(cm2� h) (De Rosa et al.2003). Therefore, the identification and quantification limits have been set approxi-mately to these values. Biochemical analysis methods requiring OPA, acetylacetoneand formaldehyde or florescamine derivatization have been developed for 5-ALAquantification in tissues, biologic matrixes, plasma, and blood samples. The LLOQfor these methods was approximately 0.001–0.4 mg=ml (Zhang et al. 2011; Namjoshiet al. 2007; De Rosa et al. 2003). The LOQ for HPLC or UPLC coupled with massspectrometry was 1� 10�3–5� 10�3 mg=ml (Zhang et al. 2011; Benton et al. 2012);for CE, it was about 100 attomoles (Lord et al. 2012). These low values were notnecessary for skin samples, as the content of 5-ALA is at least 100-fold higher in them.

The primary steps of HPLC method development were to choose the detectionsystem, appropriate column and mobile phase according to the parameters of 5-ALAin compliance with the equipment features.

5-ALA is a 3C-NH2 amino acid with very slight optical activity and very highhydrophilic properties as well as solubility in the water (50mg=ml, 25�C). No specific


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UV spectra peaks for pure 5-ALA were indicated; therefore, spectrophotometricdetection has not been deliberated. Prior to development of HPLC-ELSD methodsA and B, the analysis using derivatization experiment with OPA (Meisch, Reinle,and Wolf 1985) in our laboratory were tested. Some scientists groups have publishedapplications of fluorimetric detection-HPLC for 5-ALA assay (Oishi et al. 1996;Baliga and Kallury 2007). For our experiments, the excitation wavelength of330 nm and emission wavelength of 418 nm (Winkler and Muller-Goymann 2005)were applied. Our attempt to adjust the post-column derivatizations with OPAreagent showed problems associated with excitation=emission properties of thereagent itself. The pre-column derivatization was omitted due to the separationproblems of ALA-derivate peak from the peaks of matrix and activated OPA.

The direction of the method development was changed into a simplifiedapproach that does not need derivatization. Therefore, low temperature ELS detec-tion was chosen according to physico-chemical properties of ALA (Bunke et al. 2000).

Column has been selected according to the hydrophilic properties of the ana-lyte (5-ALA). Therefore, for method A, YMC-Pack Hydrosphere C18 column(250� 4mm, 5 mm, YMC Co. Ltd., Japan) was selected. Due to the fact that this col-umn alone does not separate the components from the skin extracts (skin matrix and5-ALA) completely, a Hichrom Hypersil H5ODS (150� 4mm, 5 mm, Hichrom, UK)column was attached for expanding total performance and prolongation of 5-ALAretention time and full separation of the matrix components from 5-ALA. ACE 3AQcolumn (150� 4mm, 3 mm, ACT, UK) was chosen for method B as highly efficientfor the analysis of hydrophilic compounds.

ELSD principle required using only volatile mobile phase modifiers; therefore,TFA was chosen for the adjustment of mobile phase pH. Purified water was chosenas the main component of mobile phase with or without ACN addition. The mobilephase was designed according to the retention of 5-ALA and matrix, which had to beseparated completely with no overlay. A concentration of 0.1% TFA was found to besuitable for method A, according to the retention of analyte; but the shape, height ofthe 5-ALA peak, and plate count was adjusted by adding 1–5% of ACN. OptimalACN concentration of 2% and flow rate of 0.9ml=min was selected. The retentiontime after application of optimized mobile phase was 5.5min (RSD¼ 0.13%).

There was no possibility to use other methods, such as capillary electrophoresisor HPLC-MS due to limited equipment resources while precolumn as well as postcol-umn derivatization did not display reproducible results. Therefore, it was decided todevelop a parallel method (method B) that would confirm the reliability of method Aand vice versa, by using different column and adjusting other parameters adequately.This methodology was based on two basic principles for increasing the theoretical per-formance for reverse-phase chromatography: by increasing the amount of stationaryphase (method A) and by decreasing the size of the column silica particles (method B).

Mobile phase for method B was chosen between the TFA concentrations of0.05% and 0.1% at flow rate of 0.2ml=min. The retention time of the matrix and5-ALA peak overlapped at about 4min by applying 0.1% TFA. After applicationof 0.05% TFA the retention time of 5-ALA was prolonged up to 6.2min(RSD¼ 0.13%) and both peaks were completely separated.

ELSD parameters were optimized by a signal-to-noise ratio of analyte peak.According to technical properties of Alltech 3300 detector, only the nitrogen flow


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rate and evaporator temperature control level had an influence on s=n. Therefore,the following conditions for ELSD were optimized: evaporator temperature 45�C,nitrogen flow rate 1 l=min, gain of 16 (method A); and evaporator temperature45�C, nitrogen flow rate 1.5 l=min, gain of 16 (method B).

METHOD VALIDATION

Specificity and Identification

Human skin contains two groups of compounds that may produce matrixeffect. First group with high possible contamination level is porphyrins that consistof 5–ALA single molecules that are bound into a heme structure and the secondgroup consists of other amino acids that are produced at the time of extraction formskin. Placebo, real, and blank samples were analyzed with HPLC to evaluate theselectivity of both methods (Figure 1).

5-ALA is a part of human cells (porphyrins consist of 5-ALA chains boundwith each other) (Di Venosa et al. 2006); therefore, the hypothesis of 5-ALA relax-ation from skin samples in the extraction process was formulated. The hypothesiswas denied with chromatographic analysis of placebo samples and their comparisonwith the standard solutions (Figure 1).

The system performance was relevant to conform the specificity of single ana-lyte HPLC, because the whole range of impurities (the known impurities and theunknown ones) must be assessed. The plate count has been established by obtainingthe average from 3 concentration levels. It was found to be 16095 for method A and8755 for method B. The performance of both systems is suitable for 5-ALA separ-ation from the matrix and unexpected impurities; and is compatible with theapproach of system suitability parameters (not less than 2000) (Vidya Sagar et al.2011). As it is obvious from Figure 1, no 5-ALA peaks were recognized in placeboand blank samples. The peaks of 5-ALA occurred in real samples and were compat-ible with the peak obtained from the standard reference solution.

Indiscretion of the overlaid peak shape was confirmed by PDA analysis in thewavelength interval from 200 nm to 500 nm. No absorption peaks in UV visible spec-tra was found at retention time of 5-ALA peak in both methods; therefore, spectralpurity of the peak was confirmed. Overall, the system specificity has demonstratedadequate separation of the analyte and is suitable for 5-ALA assay quantification.

Figure 1. Specificity of method A and B. 1) extract from epidermis (placebo 1); 2) extract from dermis

(placebo 2); 3) carbopol gel in 0.1% TFA solution (blank); 4) real sample from epidermis; 5) real

sample from dermis; 6) standard solution of 5-ALA 200mg=ml.


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Calibration Curves, Linearity, and Lowest Limit ofQuantification (LLOQ)

Based on the optimization experiment, regression coefficient of linear cali-bration has been found to be 0.991 for method A and 0.990 for method B. Therefore,quadratic equation has been chosen over linear regression according to higher valuesof regression coefficient (adequately 0.998 for method A and 0.999 for method B).Cubic regression showed to be higher by 0.01 than a quadratic one in method Aand it was the same (0.999) in method B, but the latter one has been selected dueto fewer polynomial degrees. The visualization of graph type selection has beenshown in Figure 2, where adequate coefficients of determination for different curvetypes are listed and the curves have been displayed.

Log-log analysis showed the following coefficients of determination (r-squared)for the same data: 0.995 for method A; 0.998 for method B. Log–log curves havebeen estimated with the software Empower 2. The comparison of log-log and quad-ratic equations for both methods with adequate RSS and RSD(E) values has beendisplayed in Table 1.

Table 1 identifies lower values for sum of squares (RSS) showing the differencebetween measured response and the correlated response of the calibration andRSD(E) (the line relative deviation) for quadratic curves. Both of these errors havebeen calculated with Empower 2 while displaying calibration curves. RSS andRSD(E) values display the tightness of the model fitting to the data: the lower theyare, the better that model fits the data. Therefore, from both of these perspectives,quadratic curve is chosen as the most suitable one.

Pearson’s correlation coefficient 0.995 for method A and 0.996 for method B(sig.< 0.05) has shown that there is a very strong relation between the concentrationand the signal area (mV).

The findings agreed to the estimation that linear regression is less appropriateto ELS detection than quadratic and log-log ones (Miller and Miller 2005). Follow-ing quadratic equations have been selected: (y¼ 49.5 x2þ 26651.2x� 290320.4)(method A), (y¼ 199.75x2þ 45940x� 651925) (method B).

Figure 2. Different curve fits for both HPLC-ELSD methods with R2 values displayed.


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Lowest limits of quantification (LLOQ) have been determined based on signalto noise approach. Minimum reliable quantitative concentrations (LLOQ) have beencalculated assessing 30 mg=ml (a) samples. Therefore, the equation for LLOQ detec-tion was the following one: LLOQ¼ (a� 10)=b, where b represents the EU s=n ratio.In this case, the following LLOQ values have been quantified: 20 mg=ml (method A)and 8.4 mg=ml (method B).

Accuracy and Recovery

After 2 sample sets of 6 repeated injections in 3 levels of solutions of knownconcentrations, the accuracy for both methods was established. The accuracy datawas processed by subtracting the calculated mean from the actual value and thenapplying the modulus. The lowest accuracy and highest deviation was evaluatedfor the lowest concentration point. The accuracy met the requirements for bioanaly-tical preparations and did not exceed 15% (Smith 2010).

Real samples were omitted for recovery and accuracy evaluation, because aninfinite dose was used for penetration experiments. The penetration depends onthe saline flux homogeneity, the homogeneity of 5-ALA formulations, penetrationtime, among others; hence, it was impossible to predict the amounts of 5-ALA thatwould penetrate into the skin. As a result, accuracy testing has been performed usingthe reference samples. As it is obvious from Figure 3, the accuracy increased as the

Table 1. Comparison of curve fit equations and errors for both methods

Curve type Equation R2 RSS RSD(E)

Method A Log - log y¼ 3.55x(1.72) 0.995 3.52� 1011 5.04

Quadratic y¼ 49.5x2þ 26651.2x� 290320.4 0.998 2.19� 10�2 0.49

Method B Log - log y¼ 3.67x(1.52) 0.998 1.33� 1011 5.04

Quadratic y¼ 199.75x2þ 45940x� 651925 0.999 4.55� 10�3 0.44

R2, coefficient of determination; RSS, sum of squares; RSD(E), line relative standard deviation.

Figure 3. Accuracy for method A and B. Concentration levels: 1) 30 mg=ml, 2) 100mg=ml, 3) 170mg=ml.

Identification of visualization parameters: the box represents quartiles, the line – median, bars –

deviation. (Figure available in color online.)


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percentage distance from the true value decreased; therefore, accuracy at low levelswas significantly relevantly lower, but was still compatible to deviations listed in theguidelines for biomedical preparations. At higher concentrations, the percentagedistance from the true value decreased and in total met the interval of 4–5%. Also,the total RSD for each concentration level did not exceed 15% and met the require-ments for biomedicines and bioanalytical methods. The deviations distributed morehomogenously into both positive and negative directions in method B; therefore, themedian was closer to zero. For method A, the percentage distances were onlypositive. Higher concentration levels also produced a higher proximity to zero valueof the medium.

In comparison, recovery for other methods for other 5-ALA quantificationmethods were found to be �101.8% for OPA derivatization and electrochemicaldetection (Meisch et al. 1985), 96.9%–103.6% for OPA derivatization and fluorimetricdetection (Donnelly et al. 2006), and 104.3% for fluorescamine assay (Namjoshi et al.2007). LC-MS=MS analysis produced the accuracy 95.3–103.5% for UPLC-MS=MS(Alsarra et al. 2011) and 94.1–108% for LC-MS=MS (Zhang et al. 2011). As it isshown in Figure 3, the percentage distance was higher for lower concentration levels,but it did not exceed 15% for our HPLC-ELSDmethods. The recovery for both meth-ods was compatible to the earlier established methods and was generally in the inter-val of 102.5–108.5% for other concentration levels.

Recovery for gels containing 5-ALA was performed in terms of total amountthat was put into the formulation and onto the skin. This recovery test was performedwith real samples: 200mg of gels containing 20% of 5-ALA was added onto the skinsamples placed in Bronaugh cells. Because gels have intensive properties of losingwater and forming films (therefore, the concentration of the gels may change), concen-tration of gels (without applying them to the skin) were evaluated by dissolving 0.05 gof gel containing 20% of 5-ALA in 0.1% TFA. Recovery of gels was evaluated by add-ing up the 5-ALA amount which was found in the epidermis, the 5-ALA amountwhich was found in epidermis, and the amount of 5-ALA found in the acceptor phase(sodium chloride solution that was imitating blood flow). This sum was divided by thetotal amount of 5-ALA which was re-calculated in the gels, containing 5-ALA; there-fore, the percentage of recovered 5-ALA was calculated. This experiment was per-formed with carbopol gels and has subjected following results: 68% of 5-ALA hasbeen recovered with method A and 72% was recovered with method B. These resultsare relevantly not different from the results published by other scientists working withthis penetration technique (Kubota, Sznitowska, and Maibach 1993).

Precision

All values of the precision experiment are summarized in Table 2. Inter-dayprecision was calculated out of 2 concentration sets. Repetitions of 6 times were per-formed for intra-day precision. Intra-day precision was found to be in the interval of1–2% for method A and 0.6–3% for method B which was adequate at all concen-tration levels. The range of the RSD (%) for inter-day precision varied from1.1–2.2% for method A and 2.6–3.4% for method B. The precision of both methodswas compatible with the guidelines for bioanalytical preparations (not exceeding15%) and even chemical preparations as well (not exceeding 5%) (van Amsterdam


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at al. 2010). At medium (100 mg=ml) and high (170 mg=ml) sample concentrations, theRSD highest value for intra-day repeatability was within 2%; for inter-day repeat-ability, it was within 3%. Higher precision (lower RSD %) was characteristic forhigher concentration levels. In comparison, different precision values were identifiedfor analytic and quantification methods by other authors [e.g., 2–15% for electro-chemical detection after OPA derivatization (Meisch et al. 1985), 0.75% for capillaryelectrophoresis (Bunke et al. 2000), 0.4–1.7% for fluorescamine assay (Namjoshi et al.2007), 1.9% for UPLC-MS=MS method (Alsarra et al. 2011), and 1.2–9.3% forLC-MS=MS method (Zhang et al. 2011)]. Therefore, the precision calculated forour method did not show any relevant differences from the methods with the highestprecision and it was higher in comparison to OPA derivatization method as well.

Application of Method to Quantification of 5-ALA PenetrationEx Vivo

Both methods have been used for the quantification of penetrated 5-ALA intodermis and epidermis from different bases of semi-solid preparations.

The goal of this study was to evaluate the amount of 5-ALA that penetratesinto skin layers from different bases and to show that the both parallel methodsare equally suitable for 5-ALA identification. It was also essential to compare theamounts of 5-ALA that penetrate into the skin from the cream formulation, whichis used practically for the treatment of AK in the Hospital of LUHS, and gel formu-lations that have been designed in the department of pharmaceutical technology.

It is scientifically accepted, that higher concentrations of 5-ALA penetrated intoskin produce a better treatment for AK (De Rosa and Bentley 2000). Pharmaceutical

Table 2. Intra-day and inter-day precision for 5-ALA determination in skin samples for method A and B

Intra-day repeatability

Inter-day precisionDay 1 Day 2

Method A Concentration level 1

Mean, mg=ml 32.15 31.76 31.97

RSD (%) 1.54 2.03 1.90

Concentration level 2

Mean 105.87 103.03 104.45

RSD (%) 1.90 1.16 2.17


Mean 173.718 174.135 173.95

RSD (%) 1.31 1.10 1.14

Method B Concentration level 1

Mean 28.86 30.33 29.52

RSD (%) 1.4 3.05 3.42


Mean 101.07 96.29 99.08

RSD (%) 1.45 0.84 2.75


Mean 167.23 172.13 169.41

RSD (%) 0.84 0.62 2.77


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bases for 5-ALA incorporation for penetration experiments into skin have beendesigned in the LUHS Pharmacy (cream) and the Department of PharmaceuticalTechnology [carbopol (CP), hypromellose (HM), and methylcellulose (MC) gels,pH¼ 5]. The optimal concentration of the polymers, 1% of CP, 3% of HM, and4% of MC that must be added to form a gel, have been determined after experimentalstudies performed in LUHS (Armoskaite et al. 2012).

Ex vivo skin penetration experiments were carried out as it is listed in the SamplePreparation section. Infinite doses [200mg of bases with 20% of 5-ALA or without it(control samples)] were applied. The concentration of 20% was selected as a standard(Piacquadio et al. 2004) for passive penetration experiments. Control samples wereanalyzed while applying the same amount of gel without 5-ALA to confirm that nostructures, substances, or impurities would be identified as 5-ALA. One placebo sam-ple without any base and no 5-ALA was tested, as well, after the equilibration phase.

Control and placebo samples did not show any signs of 5-ALA penetrationinto dermis or epidermis and proved again that no 5-ALA was released from the skinitself and pharmaceutical bases at the time of extraction.

The outcome of penetration experiments with HPLC-ELSD are concluded inFigure 4.

The results identify that the best medium for 5-ALA incorporation was MC gel,the second was CP gel, and the third was HM gel. The highest amount of 5-ALApenetrated from CP gel into the epidermis, from MC gels into dermis. RSD%for experimental values of method A were detected in the interval of 5.8–10.6%;adequately, identical RSD% for method B was identified in the interval 5.7–10.7%.These values meet the guidelines for biopharmaceutical preparations.

Figure 4. Quantitative analysis of 5-ALA penetration into skin layers (dermis and epidermis) from

different pharmaceutical bases. CP – carbopol, HM – hypromellose, MC – methylcellulose.


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While applying parallel validation, it was quantified that the penetrated 5-ALAvalues of parallel methods differed by RSD%¼ 2.5–5% for cream preparations andby RSD%¼ 0.05–1.4% for gels. Therefore, both methods are equally efficient andsuitable for 5-ALA identification and quantification.

According to experimental data, it was been suggested that cream manufac-tured in the Hospital of LUHS has poorer features for the 5-ALA penetration thanhydrophilic gels (as well as the quality of treatment that is determined with this para-meter) (Juzeniene, Juzenas, and Moan 2010). Penetration from gels was 1.09–1.9times higher to the epidermis and 1.38–7.8 times higher to dermis for method A thanthe cream. The following parameter was adequately 1.16–2.03 times higher for epi-dermis and 1.34� 7.62 times higher for dermis for method B in comparison to thetransdermal penetration from the cream.

CONCLUSIONS

The gain of parallel estimated methods is suitability for simple selection of thepharmaceutical mediums, which are used practically for treatment of AK in the Hos-pital of LUHS. Two LC-ELSD methods for the measurement of 5-ALA in humanskin samples ex vivo that do not require any derivatization were established and com-pared. These simple methods proved to be specific, suitably sensitive, and have beensuccessfully applied for quantification of 5-ALA that has penetrated into=throughskin ex vivo. Both methods were equally useful for the determination of 5-ALAand had no significantly relevant differences in the validation and application resultsof the same samples; therefore, both of them may be used for quantification of5-ALA in skin samples. LC-ELSD methods were acceptable for quantification of5-ALA amounts that penetrated from samples (proven with the practical applicationexperiment). After evaluation of 4 pharmaceutical bases, gels provided higher resultson 5-ALA penetration than the LUHS cream that has been practically used in theHospital of LUHS for treating patients with AK.

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application of hplc-elsd for the quantification of 5-aminolevulinic acid after penetration into...

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