Development and Validation of a Liquid Chromatography-Mass Spectrometry Assay for Determination of Cromolyn Sodium in Skin Permeation Studies

Cromolyn sodium (CS) is a mast cell stabilizer administered to treat allergic diseases. A topical system would sustain its delivery and may be designed for treatment of atopic dermatitis. Established HPLC protocols for detection of CS are time consuming and intensive, indicating the need for a more streamlined method. This study aimed at developing and validating a sensitive and selective LC-MS method for quantifying CS in skin permeation studies that was less time and resource demanding. The optimized method involved an isocratic mobile phase (10 mM NH4HCO3, pH 8.0, 90% and ACN, 10%) at a flow rate of 0.25 mL/min. Detection involved direct MS/MS channels with m/z 467.0255 (precursor) and m/z 379.0517 (fragment) using argon as the collision gas. CS calibrants were prepared in PBS, pH 7.4, and methanol for validation (0.1–2.5 μg/mL). To ensure no skin interference, dermatomed porcine skin was mounted on Franz diffusion cells that were analyzed after 24 h. The skin layers were also separated, extracted in methanol, and analyzed using the developed method. Retention time was 1.9 min and 4.1 min in methanol and buffer, respectively. No interfering peaks were observed from the receptor and skin extracts, and linearity was established between 0.1 and 2.5 μg/mL. Interday and intraday accuracy and precision were within the acceptable limit of ±20% at the LLOQ and ±15% at other concentrations. Overall, the simplified, validated method showed sensitivity in detecting CS in skin without interference and was applied to demonstrate quantification of drug in skin following 4% cromolyn sodium gel exposure.


Introduction
Cromolyn sodium, disodium 5-[3-(2-carboxylato-4-oxochromen-5-yl)oxy-2-hydroxypropoxy]-4-oxochromene-2carboxylate (CS), is a mast cell stabilizer that has been used for the management of allergic and exercise-induced asthma, systemic mastocytosis, and has also been reported to effectively prevent allergic reactions associated with atopic dermatitis [1][2][3]. It functions to inhibit histamine and leukotriene release that act as inflammatory mediators in an allergic response caused by antigen stimulation, as well as other nonspecific triggers like exercise [4]. CS also presents as a more attractive alternative to steroids, lacking the complications associated with them, such as gastric issues and decreased immunity [2].
CS therapy for asthma is administered intranasally or orally, which requires a multiple dose regimen due to its short half-life (∼80 minutes) and low bioavailability (∼1% orally and ∼7% intranasally), causing inconsistent use among patients [2]. ese properties constitute the need for developing a transdermal product for CS in asthma patients to allow for extended release, decreasing the number of doses, thus increasing patient compliance [5]. Atopic dermatitis can cause sleeplessness and severe pruritus, and conventional therapies give less than satisfying results in relieving symptoms. Previous clinical studies have demonstrated promising results with CS as a topical agent for treatment, most notably because topical use allows for direct access to mast cells of the skin [3,6]. However, no topical formulation containing CS is commercially available till date. Our studies are thus aimed at developing an effective gel incorporating CS for treatment of atopic dermatitis, utilizing skin as the mode for its delivery. In addition, the feasibility of transdermal delivery of CS for preventative therapy in asthma will also be explored. e process of developing and optimizing any effective topical or transdermal product formula involves conducting in vitro skin permeation studies to assess the permeation profile of the drug from the prepared formulation matrices using diffusion cells of varied kinds. A prerequisite for successfully conducting skin permeation studies and obtaining meaningful data is to employ a selective and sensitive analytical tool to quantify the amount of drug in the different skin layers as well as in the receptor compartment of the diffusion cells. However, owing to the complexity of the skin composition and possibility of interference due to its components during an analytical estimation, developing such an analytical method for detection and quantification of the drug of interest can be challenging [7].
Systemic circulation and metabolism of pharmaceuticals has long been investigated, and analytical methods are developed for a wide range of use in drug detection. Some such methods investigate drug stability and separation from its degraded constituents, while others quantitatively assess drug composition of pharmaceutical formulations. ese methods can also be adapted for use in human or model system biological fluids and tissues, although drug separation from inherent biological compounds should be investigated to ensure method accuracy. In vitro permeation studies are uniquely subjected to such requirement as direct analysis of drug diffusion through skin and do not implement extensive drug extraction techniques usually used for analysis of compounds in urine or serum [8]. As a result, analytical methods employed for such studies need to be able to selectively quantify target drug from skin components, like epidermal ceramides, that leach into permeation samples [9,10].
Previous methods for quantifying amount of CS in receptor solution and skin have been developed but are either too time and resource intensive, do not extend to porcine skin use, or lack mention regarding selectivity in face of skin interference.
ere exist liquid chromatography-mass spectrometry (LC-MS) protocols for detecting CS in human plasma and urine [11][12][13], but none could be found for CS detection in the skin. High performance liquid chromatography (HPLC) methods have been developed and used for quantification of CS in cellophane [1] and synthetic membranes [14,15] which are not capable of accurately mimicking permeation of drug via animal skin [1]. Of the studies that employ HPLC analysis on animal skin, such studies investigate permeation across hairless guinea pig [16,17], hairless mouse [14], or rabbit skin [15], all of which have varying characteristics when compared to porcine skin. Differences in stratum corneum thicknesses, lipid composition, and the presence of hair can affect characteristics of permeation [18,19]. A study by Rakesh and Anoop [2] utilized spectrophotometric methods on permeation across porcine ear skin but made no mention of any skin interference we observed during our application of various HPLC-UV methods. Finally, a method of HPLC coupled with ion pairing has been established for CS quantification [16,17]. Ion pairing can pose its own issues, including an increased cost associated with the quantity of ion pairing agents and dedication of single columns for single agents, as well as an increased duration of analyses based on elevated complexity of the method, mobile phase, equilibration, and selectivity [20]. It appears then necessary to investigate the use of an LC-MS assay for detection and quantification of CS in porcine ear skin that allows for selective drug detection excluding interference and is time and resource efficient.
In the present study, a new simple, sensitive, and selective method for analyzing CS in skin permeation studies using LC-MS was developed. e present study is, thus, the first one that describes the development and validation of an LC-MS method for CS detection in skin with no interference due to the skin components.  e mass spectrometer utilized an electrospray (ESI) source operating in negative ion mode. e HPLC column used was an InfinityLab Poroshell HPH-C18 (2.1 × 150, 2.7 μm) from Agilent Technologies (Santa Clara, CA, USA).

Preparation of Calibrants and
Samples. CS calibrants were separately prepared in 1X PBS (10 mM, pH 7.4) and methanol. A solution of 1 mg/mL CS was initially prepared in these solvents and further diluted to prepare stock solutions of 100 μg/mL. e following concentrations were then prepared from the stock solutions by dilution with the respective solvents: 0.1, 0.25, 0.5, 0.75, 1, and 2.5 μg/mL. All calibrants were filtered through a 0.22-μm nylon syringe filter membrane (New Oxford, PA, USA) before analysis.
To confirm the absence of interference from the skin components, porcine ear skin was dermatomed with dermatome 75 mm (Nouvag AG, Goldlach, Switzerland) to a thickness of ∼600 μm and mounted on Franz Diffusion Cells (PermeGear, Hellertown, PA, USA) containing 1X PBS. After 24 h, the receptor solution was collected and analyzed by the optimized LC-MS method. Further, the stratum corneum was separated from the same skin sample by the technique of tape-stripping using D-Squame stripping discs (Dallas, TX, USA) [21]. Twenty adhesive tape discs were applied sequentially on the permeation area of the skin for 10 s using a constant force applicator. Tapes 1, 2, 3, 4, 5, 6-10, 11-15, and 16-20 were collected separately and placed in 5 mL methanol. e viable epidermis underlying the stratum corneum was scraped off with a pair of forceps and separated from the dermis layer. Viable epidermis and dermis were then manually minced and placed individually in 2 mL methanol. e tapes, epidermis, and dermis samples were allowed to shake at 200 rpm for 4 h (Orbi ShakerTM Jr. Benchmark, Edison, NJ, USA). ereafter, the extracts obtained were filtered using 0.22 μm nylon membrane syringe filters and analyzed using the developed LC-MS method.

LC-MS Method.
e chromatographic separation involved an isocratic mobile phase (10 mM ammonium hydrogen carbonate, pH 8.0 and acetonitrile: 9 : 1) at a flow rate of 0.25 mL/min. e column was kept at 40°C. e mass spectrometer utilized liquid nitrogen as the nebulizing gas in the negative ESI source, which was maintained at 200°C. Detection utilized direct MS/MS channels with m/z 467.0255 (precursor) and m/z 379.0517 (fragment) with argon as the collision gas. Injection volume for all calibrants and study samples was 20 μL.

Validation of LC-MS Method.
Validation of the developed method was conducted following the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines for analytical procedures with respect to selectivity, linearity and range, limit of detection (LOD) and quantification (LOQ), and interday and intraday precision and accuracy [22]. As described in section 2.3., the in vitro skin permeation studies involve the use of 1X PBS in the receptor of the Franz cells and organic solvents such as methanol for the extraction of drug from the stratum corneum and other skin layers. As calibrants individually prepared in these solvents would be involved when quantifying the drug amount in the receptor and different skin layers, the developed LC-MS method was validated for the drug solutions prepared in the respective solvents, and the absence of skin interference and selectivity of the developed method was confirmed.
2.5.1. Selectivity. Standard CS solutions prepared in PBS and methanol were evaluated to ensure no interference of endogenous skin components with the drug peak. Retention time (RT) and peak areas were taken into consideration to draw suitable inferences.

Linearity and Range.
e linearity range of calibration plots was confirmed and validated for the CS samples prepared in PBS as well as methanol over three days. For each matrix, a 6-point calibration curve covered the range of 0.1-2.5 μg/ml, with specific points at 0.1, 0.25, 0.5, 0.75, 1.0, and 2.5 µg/ml. Least squares linear regression was applied, and goodness of fit was estimated based on the calculation of Pearson's determination coefficient (R 2 ) for each plot.

Intraday and Interday Accuracy and Precision.
e developed method was assessed for interday accuracy, represented by (%) error, and interday precision, represented by (%) relative standard deviation (RSD), over 3 days for calibrants prepared in PBS as well as in methanol (range as specified in section 2.5.2). Similarly, intraday accuracy and precision were determined by analyzing and comparing replicates for all calibrants analyzed on the same day. For each day of validation, 6 replicate samples were prepared for each concentration. e peak areas were compared against a calibration curve for that day, and accuracy and reproducibility were calculated using these data. Acceptable (%) RSD and (%) error were assigned at ≤15%, or ≤20%, if at LOQ [23].

Limit of Detection and Limit of Quantification.
For determination of LOD and LOQ, signal-to-noise (s/n) ratio was taken into consideration, with LOD defined as approximate s/n ∼3 and LOQ as the lowest validated concentrations with (%) RSD and (%) error ≤20%.

Method Application.
In vitro permeation of CS from a topical gel formulation (n � 3) through dermatomed porcine ear skin was studied employing the Franz Diffusion cell set up and quantifying the drug in the different skin layers using the optimized LC-MS method. Cells with 0.64 cm 2 as the diffusion area were used. Skin was clamped between the donor and receptor compartment of the cells, such that stratum corneum layer faced upwards away from the receptor, and its temperature was maintained around 32°C. e receptor chamber consisted of 1X PBS, and the donor formulation was 100 μL of CS gel (4% w/w drug in propylene glycol with 1% w/w hydroxypropyl cellulose as gelling agent). After 24 h, the unabsorbed gel was removed from the skin using Kim wipes, and the skin surface was washed sequentially three times, each with lauryl ether sulfate and DI water. CS retained in the stratum corneum, underlying viable epidermis, and dermis was extracted using methanol following a similar protocol as described in section 2.3 and quantified using the validated LC-MS method.

Method Development.
Our initial method development focused on replicating the conditions of Lin et al., using reversed phase conditions and 10 mM ammonium acetate with 0.02% trifluoroacetic acid and methanol in a gradient [13]. ese conditions worked well with aqueous samples, Journal of Analytical Methods in Chemistry but significant ion suppression using our instrumentation was noted with samples in PBS or methanolic skin extracts. Furthermore, these conditions promoted the intermittent formation of sodium adducts, as noted by Lin et al. [10]. Previously published LC-MS assays for CS quantification relied on ionization by positive ESI [11][12][13], but the structure of this drug does not lend to easy ionization in positive mode. As noted in Figure 1(a), the free acid form of this drug contains two carboxylic acid groups, which would readily ionize in negative mode. As such, our method was built around the exploitation of the natural tendency for negative ion formation. e precursor m/z 467 fragments to m/z 379 have been shown in Figure 1(b). Solubility of the CS salt as well as negative ion formation can be facilitated by higher pH conditions; thus, an HPH-C 18 column was chosen for the separation over a traditional C 18 . Buffer strength was optimized at 10 mM with a pH of 8.0. Finally, an isocratic separation was chosen over gradient to promote sample throughput.

Method Validation.
Example chromatograms for CS in PBS and methanol are shown in Figures 2(a) and 3(a), respectively. Differences in CS retention time can be accounted for by differences in solvent strength, with CS identity confirmed by the presence of m/z 467 ⟶ 379 transition. Various blank matrix extracts (receptor, stratum corneum, viable epidermis, and dermis) as described in Section 2.3 were analyzed, using the MS1 and MS2 channels associated with CS. Based on the extracted ion chromatograms at the precursor and fragment ions (m/z 467 and 379, respectively), no interfering peaks were found in any of the matrices (see Figure 2(b) for blank receptor PBS, and 3B-E for blank stratum corneum tapes, viable epidermis, and dermis extracts in methanol). e method shows good linearity across the calibration range in both PBS and methanol, as defined by Pearson's coefficient (R 2 ) > 0.99. More specifically, the average R 2 for triplicate PBS and methanol calibrations was 1.0 ± 0.00 and 0.9971 ± 0.0019, respectively. e data for the method's intraday and interday accuracy and precision evaluation for PBS and methanol samples are summarized in Tables 1 and 2, respectively. Both intraday and interday precision and accuracy, reflected as (%) RSD and (%) error, are within the predetermined acceptable ranges in both PBS and methanol. Of note, the (%) RSD and (%) error was <15% for all concentrations except the LOQ (0.1 μg/mL), which fell below 20%. Additionally, the LOD, based on a signal-to-noise ratio of 3 : 1, was 0.05 μg/mL.

Method Application.
Following the validated LC-MS method, total average amount of CS found in the stratum corneum and viable epidermis was calculated to be 10.18 ± 2.94 μg/cm 2 and 1.47 ± 0.27 μg/cm 2 respectively, and negligible drug retention (<LOQ) was observed in the dermis. More detailed distribution of the drug amounts in the individual tapes (representing the different layers of stratum corneum), entire stratum corneum (summation of the amounts in all the tapes), and viable epidermis are shown in Figure 4. Owing to CS's physicochemical properties such as high molecular weight (512.33 Da), hydrophilicity, and ionizability (pka of 1.9), its absence or extremely low amounts in the dermis is justified and highlights and supports the need for enhancers to increase delivery and permeation of CS in transdermal skin studies [2,24,25].

Method Justification.
ere exist various methodologies already established and validated for the detection of CS in pharmaceutical formulations or biological fluids, as summarized in Table 3. Some methods were designed and validated for CS detection in commonly prescribed nasal or ophthalmic pharmaceutical formulations, with interest in developing stability-indicating methods designed to separate CS from its degradation products [26][27][28][29], coadministered drugs [1,30,31], or both [32]. Few methods were only concerned with quantitation of CS present in formulation [33][34][35][36]. Some of these methods lack sufficient sensitivity for detecting lower quantities of CS present in skin permeation studies regarding earlier receptor time points and drug extracted from collected tapes or skin layers and would not provide accurate results. On the other hand, some of these methods had a small range of validated concentrations, which would not be ideal for CS determination in large quantities (that can also be observed in receptor solutions or skin layers) without dilution, making it a time and resource intensive analysis. Other spectroscopic [37] and HPLC-UV [38] methodologies designed to quantitate CS levels in formulation are described, but not much information is available on their development and validation.
Additional works have presented methods for determining CS quantity in biological fluids like aqueous humor, urine, serum, or plasma, often supporting their validation by confirming drug content from stock solutions or formulations. Moreira et al. were able to establish an effective method for trace detection of CS, but the authors concluded their approach would not be ideal for detection where CS is the primary component due to their method's high variation in accuracy and precision [39], such as for use in in vitro permeation studies evaluating CS delivery. Similarly, some of these methods used on equine urine [40], human serum [41], or human plasma [42] focus on quantitation of smaller amounts of CS, where method validation does not extend to high enough drug concentrations for convenient usage in permeation experiments without having to dilute the large number of samples. For methods where sensitivity is appropriate for permeation study application, added steps are required for analysis. Leavitt et al. [33], Gardner [43], and Aswania et al. [44] use protocols that employ more extensive separation, extraction, and purification methods that would not only be difficult for use on skin layers but are more time and resource intensive than the method reported by us.
Importantly, none of the discussed methods detailed in Table 3, regardless of sensitivity, have been evaluated for their use on skin and do not investigate selectivity of their methodology for CS detection against skin component interference either. Endogenous compounds from skin, such as lipids, leach into in vitro permeation study samples and     through human cadaver skin [45], showing successful drug separation from skin leachables. While there are methods that have been validated for use in permeation studies, only two have been developed for CS detection in skin. An ionpaired HPLC method has been used in iontophoretic CS delivery across hairless guinea pig skin but has not been  Journal of Analytical Methods in Chemistry evaluated for quantitating drug selectively in porcine ear skin and from its interfering skin components [16,17]. Ionpaired HPLC methods are also more time and resource consuming [20], indicating the need for a simpler method. A spectrophotometric method has also been used to detect CS content in in vitro permeations studies across porcine ear skin but does not establish its validated parameters and lacks mention and investigation of CS separation from skin interference [2]. e simple validated method reported in this paper is then the first to quantify CS sensitively and  T1  T2  T3  T4  T5  T6-10  T11-15  T16-20 Total Stratum Corneum Viable Epidermis

Conclusions
A negative mode ESI-LC-MS assay for quantification of CS in PBS and methanol has been developed and validated. e precision and accuracy for all points in the calibration curve, in the range of 0.1-2.5 μg/mL, fell within acceptable limits (<15%; <20% at LOQ) for both intraday and interday experiments. e use of the mass spectrometric detection helps eliminate interference from the skin matrix by monitoring direct channels associated with the precursor (m/z 467) and major fragment ion (m/z 379). Use of the HPH-C 18 column allows for higher buffer pH conditions than a conventional C 18 column, helping promote ESI conditions conducive for the acidic cromolyn analyte. Finally, the method was successfully applied to investigate the permeation of CS into the skin from a topical gel. Further, the validated method would find application in studies investigating the percutaneous permeation of CS from different topical and transdermal formulation matrices, designed with the perspective of enhancing its permeation, into and across skin.

Data Availability
e data used to support the findings of this study are included within the article in the form of tables and figures. Additional information can be provided by the corresponding author as needed.

Conflicts of Interest
e authors declare there are no conflicts of interest regarding the research or authorship of this manuscript. Linearity was validated at too small of a range for skin permeation studies and lacked analysis of skin interference Radulovic et al. (1994) [35] Journal of Analytical Methods in Chemistry 9