ARTICLE pubs.acs.org/ac
Application of Liquid Chromatography-Direct-Electron Ionization-MS in an in Vitro Dermal Absorption Study: Quantitative Determination of trans-Cinnamaldehyde Achille Cappiello,*,† Giorgio Famiglini,† Veronica Termopoli,† Helga Trufelli,† Raniero Zazzeroni,‡ Sandrine Jacquoilleot,‡ Lucia Radici,† and Ouarda Saib‡ † ‡
LC-MS Laboratory, DiSTeVA, University of Urbino, Urbino, Italy Safety and Environmental Assurance Centre, Unilever, Bedford, MK44 1LQ, United Kingdom ABSTRACT: We propose a new analytical approach, based on liquid chromatography (LC) coupled to electron ionization mass spectrometry (EI-MS), using a Direct-EI interface, for dermal absorption evaluation studies. Penetration through the skin of a given compound is evaluated by means of in vitro assays using diffusion cells. Currently, the most popular approach for the analysis of skin and fluid samples is LC coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). However, this technique is largely affected by sample matrix interferences that heavily affect quantitative evaluation. LC-Direct-EI-MS is not affected by matrix interference and produces accurate quantitative data in a wide range of concentrations. Trans-cinnamaldehyde was chosen as test substance and applied in a suitable dosing vehicle on dermatomed human skin sections. This compound was then quantified in aliquots of receptor solution, skin extract, cell wash, skin wash, carbon filter extract, cotton swab extract, and tape strip digest. On column limits of detection (LOD) and limits of quantitation (LOQ) of 0.1 and 0.5 ng/μL, respectively, were achieved. Calibration showed satisfactory linearity and precision for the concentration range of interest. Matrix effects (ME) were evaluated for all sample types, demonstrating the absence of both signal enhancement and signal suppression. The Direct-EI absorption profile was compared with that obtained with liquid scintillation counting (LSC), a recognized ME free approach. A good correlation was found with all samples and for the overall recovery of the dosed substance.
T
he penetration of topically applied substances on the skin is of special interest for the development and optimization of cosmetic products. Dermal absorption of a given compound can be evaluated by means of in vivo or in vitro assays.1,2 With regard to the assessment of dermal absorption and percutaneous penetration of cosmetic formulations, in vitro studies are recommended for ethical reasons and sample throughput:3 in vitro assays can be used equally well with animal and human skin; they allow the replicate measurements from the same or a number of different subjects; they do not need live animals and are suitable for nonradiolabeled test substances, which are extensively metabolized. The main limitation associated with an in vitro study is that conditions of peripheral blood flow may not be fully reproduced. However, dermal absorption is primarily a passive process, and studies undertaken using appropriate experimental parameters have demonstrated the usefulness of this method.4 6 In a typical in vitro dermal absorption study, the dose sample is applied to the surface of the excised skin, which is mounted in a diffusion cell (Figure 1A).7 The underside of the skin is in contact with a flowing receptor solution, to ensure sink conditions and to maintain a level of hydration. The test preparation is left on the surface of the skin for a determined period of time base on the expected human exposure. Dermal absorption evaluation is then performed through the quantitative investigation of all sample types for both tissue flux and total mass balance. In vitro dermal absorption of elemental species is normally assessed by atomic absorption spectrometry (AAS) and inductively r 2011 American Chemical Society
coupled plasma mass spectroscopy (ICPMS).8,9 Traditional analytical methods for determining the in vitro absorption profile of organic compounds include liquid scintillation counting (LSC) for radiolabled compounds,3,10 12 HPLC-UV, and hyphenated chromatographic techniques (GC/MS and LC-MS).3,13 16 Due to the recent improvements of ESI-MS/MS, this technique is now playing an increasing role for the analysis of active ingredients in tissue extracts.13,17 19 However, an important issue in the quantitative analysis using LC-MS is its susceptibility to matrix effects. These phenomena are observed when the analyte response is suppressed or enhanced due to coeluting matrix components.20 Matrix effects (ME) can heavily affect the reproducibility, linearity, and accuracy of the method leading to an erroneous quantitation.21,22 As a consequence, actions during method development must be taken prior to MS detection to minimize their impact.23 Sample cleanup procedures and more efficient chromatographic separations could reduce the introduction of interfering compounds into the MS system. However, they are sometimes laborious and time-consuming, and subjected to analyte losses. The use of stable isotope-labeled internal standards (SIL-IS) has also been attempted. However, it is worth pointing out that labeled standards are expensive and not always commercially available for all compounds. Many authors highlighted the occurrence of ME in bioanalytical, environmental, Received: July 18, 2011 Accepted: October 4, 2011 Published: October 04, 2011 8537
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Figure 1. Flow through diffusion cell (A) and in vitro skin penetration experimental setup (B).
and food analysis.24 26 In our own experience, these phenomena are mainly observed when dealing with skin digests and receptor solution due to the complexity of their chemical makeup and high concentration of salt and antibiotics. The objective of the present work was the evaluation of Directelectron ionization (EI) as an alternative LC-MS interface for the development of a ME free LC-MS approach in support of in vitro dermal absorption studies. The method relied on then use of LC coupled to an EI-MS system. The LC-Direct-EI-MS interface is a simple device in which the liquid flow coming from a nanoLC system enters directly into the EI ion source of a mass spectrometer.27 An extensive discussion on the role of EI in LC-MS interfacing and on its performance can be found in literature.28 31 Briefly, the LC-DirectEI-MS offers a valid alternative to atmospheric pressure ionization (API) and preserves the advantages of EI when coupled to an LC separation. These advantages can be summarized in three crucial aspects: automated library identification; identification of unknown compounds due to EI extensive fragment information; inertness to coeluted matrix interferences due to very unlikely occurrence of ion ion and ion molecule interactions in the EI gas-phase environment. In the present paper, these key features of Direct-EI-MS were tested in an in vitro skin absorption study for trans-cinnamaldehyde.
This molecule was chosen as test substance for its widespread use within the cosmetic industry. This compound is investigated by ESILC-MS showing a significant signal suppression due to matrix interferences resulting in an erroneous quantitation.
’ EXPERIMENTAL SECTION Reagents. LC grade solvents and chemicals were purchased from VWR International (Milan, Italy). Milli-Q water was obtained from a Millipore direct-Q 3 UV purification system (Millipore Corp., Bedford, MA). Working solution of trans-cinnamaldehyde (CAS number: 104-55-2, MW: 132) was prepared at 2.5% (w/v) in acetone/olive oil (4:1, v/v). The receptor solution was prepared by dissolving 5 phosphate buffered saline tablets and adding 10 mL of amphotericin B [250 μg mL 1], 10 mL of penicillin/streptomycin [10 000 IU mL 1/10 mg mL 1] and 50 mL of newborn calf serum (NCS). In Vitro Dermal Absorption Experiment. Penetration through the skin, as well as the absorption into different skin compartments of a compound, can be evaluated by means of in vitro assays that are based on diffusion cells using a wide variety of animal and human skin samples. The in vitro absorption studies were based on the “Guidance document for the conduct of skin absorption studies” of 8538
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Figure 2. DirectEI mass spectrum of trans-cinnamaldehyde is shown in the upper trace and is compared with the standard NIST EI library spectrum shown in the lower trace.
the Organisation for Economic Co-operation and Development (OECD).32 The main principle of diffusion cells is that an isolated section of skin is supported stratum corneum face up above a receptor chamber containing a receptor fluid (Figure 1A). The test substance is applied in an appropriate formulation on the skin sample. The applied compound penetrates through the skin section and is recovered in the receptor fluid which, when sampled regularly, allows an absorption profile of the applied compound to be constructed. The amount of penetrated compound in the layers of the stratum corneum is usually measured by the tape stripping technique which consists of repeated application and removal of adhesive tape on the surface of the skin, whereby consecutive layers of stratum corneum are sampled (Figure 1B). Ethical approval has been received from the Internal Research Ethics Committee for the use of human skin in this study. Frozen dermatomed human skin was supplied by TCS CellWorks. The skin was allowed to thaw thoroughly at room temperature or in a refrigerator before use. The cells were left to equilibrate with receptor solution at 37 °C for approximately 1 2 h. Four cells were dosed with 20 μL of the test preparation. The cells were capped with a chamber containing a carbon filter to trap any of the test item volatilizing off the skin surface. Treatment of in Vitro Skin Penetration Samples. The following samples were analyzed by LC-Direct-EI-MS: homogenized skin samples, receptor fluid, cell wash, skin wash, tape strip extract, carbon filter extract, and cotton swab extract. During the experiments of ME evaluation, blank samples were fortified with trans-cinnamaldehyde to a final concentration of 0.2 ng/ μL. Receptor Solution Samples. Receptor solution was collected hourly from each cell from t = 0 to t = 24 h. Prior to analysis, receptor fluid was subjected to a solid-phase extraction (SPE) using a 3 mL cartridge packed with 200 mg of ODS-C18 stationary phase (Agilent Technologies Inc., Santa Clara, CA). The cartridges were conditioned by passing 10 mL of methanol and 10 mL of water. Receptor fluid (4 mL) was forced through the cartridge at a flow rate ranging from 12 to 15 mL/min. Before elution, the cartridges were dried under vacuum for 10 min. After that, the analytes were eluted with 4 mL of methanol and finally diluted with 4 mL of water. The percentage of recovery was of 100% with a RSD% of 4%. Filter Extract. The traps were removed from each cell, and the carbon filters were placed into a 20 mL glass vial containing 10 mL of methanol. Each vial was capped and vortexed for 1 h. The carbon filter was removed, and 10 mL of ultrapure water was added to the solution. An aliquot was filtered and transferred in a glass vial.
Skin Wash. The skin was rinsed with ten 0.5 mL aliquots of Palmolive liquid hand wash diluted with water (2%, v/v). The soapy water was aspirated with a pipet and collected in a 10 mL volumetric flask. The volume of each volumetric flask was made up with methanol. One milliliter of each sample was diluted to 5 mL in a volumetric flask with methanol/water (50:50, v/v) Swab Extract. The skin was blotted dry using a cotton swab which was extracted with 20 mL of methanol/water (50/50, v/v) and vortex mixed for 2 min. Prior to the LC-Direct-EI-MS analysis, the extracts were filtered with a 0.2 μm syringe filter (Whatman syringe filter PURADISC 4, 0.2 μM, PTFE). Cell Wash. The skin was then removed from the cell, and the cell was soaked in 50 mL of 50% (v/v) aqueous methanol with the traps at room temperature overnight. Tape Strip Extract. The samples of inner skin were tapestripped with 10 disks of D-SQUAME. The disks were placed separately in a 15 mL centrifuge tube and dissolved with 1 mL of ethanol/isopropanol (50:50, v/v). Then, 1 mL of 0.05% formic acid in water was added to the mix. This solution was centrifuged for 20 min at 4000 rpm, and the supernatant was filtered (Whatman syringe filter PURADISC 4, 0.2 μM, PTFE). Outer Skin Extract. The flange skin (outer skin, OS) was trimmed off and divided into four pieces. Each piece was placed in a FastPrep Matrix F tube between two ceramic balls. One milliliter of 50% (v/v) aqueous methanol was added to each tube, and the skin was homogenized using the FastPrep for 4 30 s at 6.5 m/s. The extract was filtered through a 0.2 μm filter (Whatman syringe filter PURADISC 4, 0.2 μM, PTFE). All the extracts were combined and collected into a glass vial. Inner Skin Extract. Each piece of inner skin was placed in a FastPrep tube with 1 mL of methanol/water (50:50 (v/v)) homogenized by FastPrep method (4 30 s at 6.5 m/s) and filtered (Whatman syringe filter PURADISC 4, 0.2 μM, PTFE). The extract was then transferred into a glass vial. LC-Direct-EI-MS Conditions. Chromatographic separations were performed on an Agilent 1100 series nanoHPLC system (Agilent Technologies Inc., Santa Clara, CA) using an Agilent Zorbax SB-C18 column (150 mm 75 μm i.d., 3.5 μm particle size). The injection volume was 500 nL, and the flow rate was set at 400 nL/min. The mobile phase was composed of water and acetonitrile. The percentage of acetonitrile was changed from 0% to 100% in 15 min. An Agilent 5975B Inert MSD single quadrupole mass spectrometer (Agilent Technologies Inc., Santa Clara, CA) equipped with a Direct-EI interface was used as system of detection.31 The temperature of the ion source was kept at 350 °C. Data acquisition during the chromatographic separation was carried out in selected ion monitoring mode (SIM) on the following 8539
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characteristic ions of trans-cinnamaldehyde: 103, 131, and 132 m/z. Dwell time was set at 450 ms (cycle/s 0.82). Liquid Scintillation Counting. Liquid scintillation counting technique is traditionally used to determine the distribution profile of a radiolabeled compound within the skin. Ten milliliters of IrgaSafe Plus Scintillation cocktail was directly added to aliquots of receptor solution samples, cell wash, skin wash, carbon filter, and cotton swab extracts. The skin and tape strips were digested in a 2 mL of tissue solubilizer Soluene-350 at approximately 70 °C overnight. HionicFluor (10 mL) was added to each sample, and the samples were counted. The amount of penetrated radiolabeled compound was estimated from the counts of radioactivity present in the samples.
’ RESULTS AND DISCUSSION Direct-EI Instrumental Performance. Prior to analysis of real samples, experiments on trans-cinnamaldehyde standard solutions were carried out in order to investigate the qualitative and quantitative performance of the Direct-EI interface. Spectrum acquisition was first carried out in full scan mode to investigate the Direct-EI capability of generating high quality EI spectra of the test substance. The quality of the recorded spectra was expressed as the measure of the degree of overlap with the reference spectra reported in the National Institute of Standards and Technology (NIST) electronic library. In Figure 2, the Direct-EI mass spectrum of trans-cinnamaldehyde is shown in the upper trace and is compared with the standard NIST EI
Table 1. ME Evaluation sample
a
ME ( RSD%a
cell wash
99 ( 3
skin wash
104 ( 5
swab tape strip
99 ( 2 106 ( 5
receptor fluid
100 ( 4
skin samples
99 ( 5
3 lots (n = 3).
library spectrum shown in the lower trace. This spectrum was collected at a concentration of 0.5 ng/μL (total injected amount, 250 pg) corresponding to the limits of detection (LOD) in scan mode, with the aim of investigating the library match results in the presence of a high background noise. A value of 937 was obtained for both matching factor and reversed matching factor, thus indicating a satisfactory identification of the target substance. Instrumental quantitative performance was evaluated operating in SIM mode using the program reported in the Experimental Section. Validation data were expressed in terms of LOD, limits of quantitation (LOQ), linearity, and precision. The instrumental LOD and LOQ were expressed as the analyte concentrations which gave, respectively, a signal-to-noise ratio (S/N) of 3/1 and of 10/1. The LOD was of 0.1 ng/μL, corresponding to an absolute injected amount of 50 pg, while the LOQ was of 0.5 ng/μL (total injected amount, 250 pg). These values were satisfactory for the quantitative purposes of the in vitro dermal absorption experiments. Response linearity was evaluated by the construction of a five point calibration curve. Each point was the mean of five replicates. Linear regression analysis was used to calculate the slope, intercept, and correlation coefficient (R2). Linearity was in the range of 0.1 100 ng/μL with R2 of 0.9998. Intraday and interday precision were calculated by injecting a 5 ng/μL trans-cinnamaldehyde standard solution. Intraday precision was calculated from five replicates in one working day, while interday precision was evaluated over five working days (five replicates per day). Intraday and interday precision results were 3% and 4% RSD, respectively. Matrix Effects Evaluation. As reported in the introduction section, when dealing with complex matrixes, such as skin extracts and receptor fluid samples, the occurrence of ME needs to be carefully investigated to ensure proper method precision, accuracy, and sensitivity. The Food and Drug Administration (FDA) guidelines on bioanalytical analysis explicitly require the evaluation of ME.32,33 However, there is no agreement on how this should be done. In the present work, ME was evaluated using the postextraction addition method.21,22 It is based on the comparison of the analyte signal in a standard solution and in a matrix-matched standard. This approach prevents any recovery miscalculation and
Figure 3. Distribution of trans-cinnamaldehyde (% applied dose) at 4 and 24 h post dose following topical application to dermatomed human skin: (A) LSC; (B) LC-Direct-EI-MS. 8540
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Analytical Chemistry focuses only to ME. Matrix-matched standards were prepared with blank samples of cell wash, skin wash, extracts from receptor solution, homogenized skin, tape strip, and cotton swab. Due to clogging problems related to the nano-LC equipment, receptor solution was subjected to SPE prior to analysis. As reported in the Experimental Section, all the samples were fortified with transcinnamaldehyde to get a final concentration of 0.2 ng/μL. This corresponds to an absolute injected amount of 100 pg, a value which is twice the LOD of the LC-Direct-EI-MS system. These experiments were performed at such low concentration in order to maximize the detrimental effect of the interferencing compounds present in the real samples. As proposed by Matuszewski and coworkers, the ME was calculated in percent (ME%) as the ratio between the average peak area of the matrix matched standard (five replicates) and the average peak area of the standard (five replicates) multiplied by 100. In this context, a result higher than 100% indicates signal enhancement, whereas a value lower than 100% indicates signal suppression. Three different lots of each dermal absorption sample were subjected to the ME evaluation procedure. As shown in Table 1, the response of trans-cinnamaldehyde was not affected by ME, even in the presence of complex matrixes such as receptor fluid SPE extract and skin. This result is related to the gasphase ionization that occurs in EI, which is independent by the adverse influence of interferencing compounds.28,30,31 This represents a significative advantage over LC-ESI-MS/MS where a chemical ionization process in the liquid phase makes this experimental approach severely affected by this phenomenon.20,23,34 In fact, a strong signal suppression was observed when analyzing matrix matched standards of skin and receptor solution (ME lower than 15%, which corresponds to a signal suppression higher than 85%). Because of the Direct-EI inertness toward ME, the construction of matrix matched calibration curves has been be avoided, thus simplifying the entire method validation procedure. In Vitro Skin Penetration of trans-Cinnamaldehyde. All the samples coming from the dermal absorption experiment were analyzed by LC-Direct-EI-MS in order to evaluate the transcinnamaldehyde total distribution and percentage of recovery. Approximately 8.5% of the applied dose was found in receptor fluid after a 4 h application to human skin (Figure 3A). The skin and cell wash removed about 35.4% and 8.9% of the dose, respectively. A 6.7% of the dose remained in the inner skin, while about 4.1% was found in the outer skin. About 29.4% was detected in the extracted cotton swab, while 2.7% was found in tape strip. The dose (0.6%) was recovered in the filter. The total recovery of the applied dose was about 96%. At 24 h, after the application of trans-cinnamaldehyde to human skin, approximately 16.8% of the dose was detected in the receptor fluid (Figure 3A). The skin and cell wash removed about 17.0% and 10.5% of the dose. Following the wash, the inner skin contained 1.8% of the applied dose, while 1.1% was detected in the outer skin. Approximately 5.2% was detected in the swab, while 2.7% was found in the strips. The applied dose (9.2%) was quantified in the filter. The total recovery after 24 h was 64.3%. The DirectEI-MS data were compared with those obtained with LSC, a ME free technique which is widely used to support in vitro skin penetration studies for the detection and quantification of radiolabeled chemicals. This comparison showed a good correlation with the overall recovery of the dosed substance, meaning that no ME are observed (Figure 3). The low recoveries after 24 h (66.3% and 64.3% with LSC and LC-DirectEI-MS, respectively) of exposure and the increase in the amount of trans-cinnamaldehyde recovered from
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the charcoal filters with time could suggest that a significant amount of the test item could have volatilized off over time. However, the recovery of the test material in the terminal skin surface washes also significantly decreased with time. This would then indicate that the loss of trans-cinnamaldehyde was related to a strong binding of the test compound to the skin matrix. These species are believed to be strongly bound to the complex protein mixture and therefore could not be extracted under the experimental conditions. A satisfactory correlation between LC-Direct-EI-MS and LSC was achieved also in the recovery of each sample, as shown in Figure 3A,B.
’ CONCLUSIONS Analyte signal suppression originating from matrix components is a common phenomenon when ESI-MS/MS detection is applied to in vitro dermal absorption studies. The occurrence of these ME can severely compromise the results of the quantitative LC-MS data, leading to poor accuracy of measurement. For this reason, elimination and/or compensation for ME is a crucial component of the LC-MS method validation. In this study, the dermal absorption profile of trans-cinnamaldehyde was evaluated using Direct-EI-MS. The results presented in this report show that the distribution analysis of trans-cinnamaldehyde in the different compartments by Direct LC-EI-MS was successful. The compound could clearly be detected in all types of generated sample (skin samples, receptor solution samples, cell washes, terminated surface skin washes, filter and swab extracts, tape strip extracts, etc.) with a high level of sensitivity. LC-Direct-EI-MS was not affected by matrix interference and produced accurate quantitative data in a wide range of concentrations. It demonstrated the absence of both signal enhancement and signal suppression. A good correlation with previous studies was found with all samples and also for the overall recovery of the dosed substance. This is the first application of LC-EI-MS in the evaluation of in vitro skin penetration experiments. The results obtained on skin and receptor solution demonstrated that the Direct-EI-MS, using gas-phase ionization, allowed the analysis of the test substance regardless of the presence of coeluted interferences, inevitably transferred into the system from the matrix. The Direct-EI-MS absorption profile of trans-cinnamaldehyde was in accordance to that obtained by LSC. Due to the Direct-EI inertness toward ME, the proposed method showed an improved accuracy and faster and simpler sample preparation procedure. This could become a viable tool in support of in vitro skin absorption experiments. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: +39 0722 303344. Fax: +39 0722 303346.
’ ACKNOWLEDGMENT The authors acknowledge Agilent Technologies for providing the analytical instrumentation. ’ REFERENCES (1) Bronaugh, R. L.; Wester, R. C.; Bucks, D.; Maibach, H. I.; Sarason, R. Food Chem. Toxicol. 1990, 28, 369–373. (2) Bronaugh, R. L.; Maibach, H. I. In vitro percutaneous absorption: principles, fundamentals, and applications; CRC Press: Boca Raton, FL, 1991. 8541
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