Determination of prostaglandin analogs in cosmetic products by high performance liquid chromatography with tandem mass spectrometry

Journal of Chromatography A, 1359 (2014) 140–146

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Determination of prostaglandin analogs in cosmetic products by high performance liquid chromatography with tandem mass spectrometry James B. Wittenberg ∗ , Wanlong Zhou, Perry G. Wang, Alexander J. Krynitsky U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, HFS-717, College Park, MD 20740-3835, USA

a r t i c l e

i n f o

Article history: Received 24 March 2014 Received in revised form 1 July 2014 Accepted 13 July 2014 Available online 19 July 2014 Keywords: Prostaglandins Cosmetics QuEChERS LC-MS/MS

a b s t r a c t A method was developed and validated for the determination of 16 prostaglandin analogs in cosmetic products. The QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, Safe) liquid–liquid extraction method, typically used for pesticide residue analysis, was utilized as the sample preparation technique. The prostaglandin analogs were chromatographically separated and quantified using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Thirty-one cosmetic products were surveyed, and 13 products were determined to contain a prostaglandin analog with amounts ranging from 27.4 to 297 ␮g/g. The calculated concentrations for the cosmetic products were in a similar range when compared to the concentrations of three different prostaglandin analog-containing prescription products. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Eyelash and eyebrow enhancing cosmetic products have been increasing in demand and popularity in recent years. Some of the chemicals responsible for enhancements such as lengthening and darkening eyelashes and eyebrows are a class of compounds called prostaglandins [1]. Prostaglandins are used in many biological processes and have a wide variety of effects. A few prostaglandin analogs (bimatoprost, latanoprost, and travoprost) are used to control intraocular pressure, or treat glaucoma, through the administration of prescription eye drops [2–9]. Side effects of this treatment include conjunctival hyperemia, excessive tearing, inflammation, increased coloring of the iris, periocular skin pigmentation, as well as an increase in eyelash thickness and length [10–12]. These side effects that increase and enhance eyelashes gave reason to implement prostaglandin analogs for use in cosmetic products. The prostaglandin analogs studied, including the three used as drugs for glaucoma treatment, are structurally similar (Fig. 1). They all are comprised of a cyclopentanediol backbone with two arms: one arm containing an aromatic moiety, and the other containing a carbonyl functional group. Fortunately, they are dissimilar enough to be chromatographically separated and quantified using high performance liquid chromatography with

∗ Corresponding author. Tel.: +1 2404022206. E-mail address: [email protected] (J.B. Wittenberg). http://dx.doi.org/10.1016/j.chroma.2014.07.032 0021-9673/© 2014 Elsevier B.V. All rights reserved.

tandem mass spectrometry (HPLC-MS/MS). A few of the noted prostaglandins, along with many others, have previously been analyzed in various matrices. Many techniques, including high performance liquid chromatography paired with ultra-violet absorption (HPLC-UV) [13–16] or fluorescence detection [17,18], gas chromatography–mass spectrometry (GC/MS) [19], high performance liquid chromatography-electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) [20–29], and high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC-ACPI–MS/MS) [30,31], have been used to determine prostaglandins contained in a wide variety of matrices ranging from in vitro enzyme incubations to pharmaceuticals and natural products. In general, cosmetic product matrices are complex and can be completely different in composition for two products used for the same purpose. Some of these matrices include serums, oils, lotions, creams, mascaras, powders, lipsticks, etc. Sample preparation, therefore, can be challenging. Traditionally, cosmetic samples are prepared using conventional liquid–liquid extraction (LLE) and solid–liquid extraction (SLE) techniques [32]. While these conventional sample preparation techniques are useful when dealing with a variety of matrices, there are disadvantages such as use of solvent (large quantities and potentially carcinogenic) and time consuming multi-step processes. For this project, the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) extraction method was implemented for sample preparation. The QuEChERS method was originally developed and utilized for pesticide analysis since these samples come from a large variety of matrices [33–36].

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141

Fig. 1. Structures of the prostaglandin analogs studied.

QuEChERS was chosen for this study because it was envisioned to be a quick, easy, and cheap way to extract the relatively non-polar prostaglandin analogs from the aqueous serums and mascaras. To date, no method has been reported for the detection and quantitation of prostaglandin analogs in cosmetics. In this study an LC/MS/MS method was developed to determine 16 prostaglandin analogs contained in a survey of 31 eyelash and eyebrow enhancing cosmetic products using four stable isotopically labeled internal standards. The QuEChERS extraction method was adopted and utilized for sample preparation of the cosmetic samples without any need for further sample cleanup. To our knowledge, this would be the first account of the QuEChERS method being used for cosmetic sample preparation. This method may be used for the detection and quantitation of the noted prostaglandin analogs in a variety of matrices.

2. Material and methods 2.1. Chemicals LC-MS grade water (H2 O), methanol (MeOH), and acetonitrile (MeCN) were purchased from Fisher Scientific (Pittsburgh, PA). All prostaglandin analogs (bimatoprost (bima), bimatoprost isopropyl ester (bima IE), bimatoprost serinol amide (bima SA), bimatoprost free acid (bima FA), latanoprost (lat), latanoprost free acid (lat FA), tafluprost (taf), tafluprost ethyl amide, (taf EA) tafluprost ethyl ester (taf EE), travoprost (trav), (+)-cloprostenol (clo), (+)-cloprostenol isopropyl ester (clo IE), 17-phenyl trinor prostaglandin E2

serinol amide (17-PTPE2 SA), 17-phenyl trinor prostaglandin F2␣ methyl amide (17-PTPF2␣ MA), 17-trifluoromethylphenyl trinor prostaglandin F2␣ ethyl amide (17-CF3 PTPF2␣ MA), 16-phenoxy prostaglandin F2␣ ethyl amide (16-PPF2␣ EA)) and internal standards (bimatoprost-d4 (bima-d4 ), bimatoprost free acid-d4 (bima FA-d4 ), latanoprost-d4 (lat-d4 ), and latanoprost free acid-d4 (lat FAd4 )) were purchased from Cayman Chemical (Ann Arbor, MI). All chemicals were sold as ≥95% pure and were used without further purification. Thirty personal care products were purchased via the internet. 2.2. Instrumentation The liquid chromatography was carried out using an Acquity UPLC® (Waters, Milford, MA) consisting of a binary solvent manager and a sample manager. The sample manager was set to 4 ◦ C during operation. The separations were carried out using a 2.6 ␮m Kinetex XB-C18 column (100 mm × 2.1 mm i.d., Phenomenex, Torrance, CA) coupled to a 0.5 ␮m KrudKatcher ultra HPLC in-line filter (0.004 in i.d., Phenomenex, Torrance, CA). Elution was completed using a 20-min gradient program operating with a 0.50 mL/min flow rate with a 10 ␮L injection volume. Mobile phase A was composed of 0.1% formic acid in 95:5 H2 O:MeOH (v/v) and mobile phase B was composed of 0.1% formic acid in 5:95 H2 O:MeOH (v/v). The gradient parameters were: 0–1 min 20% B, 1–15 min 20% B to 100% B, 15–18 min 100% B, 18–19 min 100% to 20% B, 19–20 min 20% B. The LC elute was introduced to the ion source only between 7 and 12.5 min during the run using a Valco valve switch to prevent contamination.

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Table 1 Scheduled MRM parameters for quantitation and confirmation transitions for prostaglandin analogs studied. Analyte

Average mass

Precursor ion

Product ion

17-PTPE2 SA

RT (min) 7.7

459.3

Bima SA

8.2

461.3

16-PPF2␣ EA

8.4

417.3

17-PTPF2␣ MA

8.7

401.3

Bima

9.2

415.3

Bima-d4

9.2

419.3

Bima FA

9.4

388.2

Bima FA-d4

9.4

392.2

Taf EA

9.7

437.2

17-CF3 PTPF2␣ EA

10.3

483.3

Lat FA

10.7

390.2

Lat FA-d4

10.7

394.3

Clo FA-Na+

10.8

446.2

Taf EE

11.4

438.2

Bima IE

11.5

430.3

Lat

11.7

432.3

Lat-d4

11.7

436.3

Clo IE

11.8

466.2

Taf

11.9

452.2

Trav

11.9

500.2

442 442 444 444 418 418 384 384 398 398 402 402 387 387 391 391 438 438 430 430 389 389 393 393 423 423 439 439 413 413 433 433 437 437 489 467 453 453 501 501

406 92 408 317 234 270 348 317 362 317 366 321 343 193 347 197 306 232 343 385 345 147 349 147 295 219 335 289 377 131 337 379 341 383 447 321 335 261 321 249

CE (V) 23 27 19 23 15 19 15 21 19 21 19 21 −28 −34 −28 −34 15 21 31 25 −36 −44 −36 −44 −24 −24 17 23 17 41 23 15 21 19 35 17 19 25 11 19

DP (V) 81 101 56 61 96 96 76 76 76 56 76 56 −90 −45 −90 −45 86 56 161 181 −140 −60 −140 −60 −60 −85 81 81 66 76 91 91 81 86 141 91 81 81 96 106

CXP (V) 10 10 10 12 18 16 12 20 10 22 10 22 −9 −5 −9 −5 14 10 12 20 −15 −7 −15 −7 −41 −15 16 10 22 22 26 18 14 10 18 16 22 16 46 6

Note: RT, retention time; CE, collision energy; DP, declustering potential; CXP, cell exit potential. The first product ion for each analyte was used for quantitation, and the second product ion was used for confirmation.

The LC was interfaced to an AB Sciex QTrap 5500 (AB Sciex, Foster City, CA) equipped with a Turbo Ion Spray® source (electrospray). LC-MS/MS operation and data acquisition were controlled by the AB Sciex Analyst software version 1.6. Quantitation was completed using the AB Sciex MultiQuant software version 2.1.1. The ion spray voltage was set to 5000 V. The turbo heater was maintained at 400 ◦ C. Nitrogen was used as the curtain gas and collision gas. The curtain gas, ion source gas 1, and ion source gas 2 were set to pressures of 30, 40, and 50 psi, respectively. The entrance potential was set to 10 V for all transitions. The collision gas was set to “Medium”. The mass spectrometer was operated in scheduled selected reaction monitoring (SRM) mode. All the parameters listed above were used for both positive and negative mode acquisitions. Other parameters (declustering potential, collision energy, and cell exit potential) were optimized for each compound and are listed in Table 1. 2.3. Preparation of standard solutions A stock solution containing 5 ␮g/mL of bima, taf EA, and 17-PTPF2␣ MA; 10 ␮g/mL of bima IE, bima SA, clo IE, lat, trav, and 16-PPF2␣ EA; and 20 ␮g/mL of taf, taf EE, 17-PTPE2 SA, 17CF3 PTPF2␣ EA, bima FA, lat FA, and clo FA-Na+ in 50:50 H2 O:MeOH (v/v) was prepared in a volumetric flask and stored at −20 ◦ C. A stock solution of internal standard mixture containing 2 ␮g/mL of bima-d4 , lat-d4 , bima FA-d4 , and lat FA-d4 in 50:50 H2 O:MeOH

(v/v) was prepared separately in a volumetric flask and stored at −20 ◦ C. Eight calibration solutions were prepared using the stock solution. The concentration ranges for the standard solutions were: 0.25–50 ng/mL for bima, taf EA, and 17-PTPF2␣ MA; 0.5–100 ng/mL for bima IE, bima SA, clo IE, lat, trav, and 16-PPF2␣ EA; and 1–200 ng/mL for taf, taf EE, 17-PTPE2 SA, 17-CF3 PTPF2␣ EA, bima FA, lat FA, and clo FA-Na+ . A constant concentration of 10 ng/mL of the internal standard mixture was added to each standard solution. 2.4. Sample preparation The majority of the cosmetic samples (including all samples containing prostaglandins) were water-based solutions and sample preparation was relatively straightforward. Stock solutions of each cosmetic sample were prepared by weighing a known amount sample (approx. 50–100 mg) and dissolving it in 50:50 H2 O:MeCN (v/v) in a volumetric flask. Any cosmetic sample that did not completely dissolve (e.g., mascaras) was sonicated for 10 min before sample preparation. The Bond Elut QuEChERS extraction kit (Agilent Technologies, Santa Clara, CA) was utilized for this project and samples were prepared in triplicate. A 100 ␮L aliquot of a stock solution of each sample was placed into a 50 mL centrifuge tube with 10 mL H2 O, 10 mL MeCN, and 100 ␮L of the internal standard stock solution and shaken for 1 min. A salt mixture (4 g NaCl, 1 g MgSO4 ) was added to the tube to induce phase separation and then mixed on

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a Vortex Maxi Mixi mixer (Thermo Scientific, Waltham, MA) for 3 min. The sample was then centrifuged at 6000 rpm for 5 min. A 500 ␮L aliquot from the organic phase was diluted with 500 ␮L of H2 O, vortexed, and filtered through a 0.2 ␮m PTFE Acrodisc CR 13 filter (Pall Life Sciences, Port Washington, NY) directly into an autosampler vial for LC-MS/MS injection. 3. Results and discussion 3.1. Mass spectrometry optimization All parameters were optimized by infusing a 1 ␮g/mL solution of each prostaglandin analog at a flow rate of 10 ␮L/min. Positive ionization mode was used for all but the four free acids (bima FA, bima FA-d4 , lat FA, lat FA-d4 , and clo FA-Na+ ), which were optimized using negative ionization mode. Both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were tested as ionization sources. APCI was initially chosen as the ionization source because a significant number of the prostaglandin analog parent ions observed for ESI were sodium adducts [M + Na]+ . As an attempt to break up these sodium adducts, ammonium formate (NH4 HCO2 ) was added to the mobile phase with no positive results. While APCI resulted in less adducts forming, ESI provided greater sensitivity to the majority of compounds (e.g., 4-fold for Bima IE and >70-fold for Bima FA), and thus was ultimately used for this study. To avoid using the [M + Na]+ adducts as the precursor ions, the ions indicating loss of H2 O were instead used. As seen in Fig. 1, all the compounds studied contain multiple –OH groups and readily lose H2 O during ionization. 3.2. Liquid chromatography optimization A number of C18 columns were tested. Initially, APCI was chosen as the ionization source. When using APCI it is important to use a high flow-rate (≥0.5 mL/min) since sensitivity is flowdependent. Therefore, a long, large-bore column (Kinetex 5 ␮m XB-C18 250 mm × 4.6 mm, Phenomenex, Torrance, CA) was initially chosen. Unfortunately, peak shape and separation were not ideal. The switch to ESI allowed for lower flow rates and the possibility to use a sub-2 ␮m UPLC column (Acquity UPLC BEH Shield RP18 1.7 ␮m 100 mm × 2.1 mm, Waters, Milford, MA) which did produce acceptable peak shape and separation. However, the ability to utilize a single LC method for both ESI and APCI was intriguing. A Kinetex 2.6 ␮m XB-C18 100 mm × 2.1 mm column was chosen for the final method since it permits higher flow rates with lower back pressures and produces similar results to the chromatography observed with sub-2 ␮m UPLC columns. All 16 prostaglandin analogs eluted between 7.5 and 12 min during the 20 min run (Fig. 2a). A mobile phase composed of H2 O:MeOH:1%FA was used. The addition of 1% formic acid was beneficial to the positive ionization while not significantly decreasing the negative ionization, thus the mobile phase was used in both positive and negative ionization modes to allow for a single chromatographic run. MeOH was chosen initially over MeCN because it is more conducive to the ionization process in APCI. The mobile phase was not changed once ESI was implemented as there was no significant change in ionization due to mobile phase change. Also, using the H2 O:MeOH:1%FA gradient allows for either ESI or APCI to be utilized in separate runs with the same LC method. 3.3. Method validation An eight-point calibration curve was created for each prostaglandin analog in the concentration ranges from 0.25 to 50 ng/mL for bima, taf EA, and 17-PTPF2␣ MA; 0.5 to 100 ng/mL for bima IE, bima SA, clo IE, lat, trav, and 16-PPF2␣ EA; and 1 to

143

200 ng/mL for taf, taf EE, 17-PTPE2 SA, 17-CF3 PTPF2␣ EA, bima FA, lat FA, and clo FA-Na+ . The lower limit of quantitation (LLOQ) was established to be the lowest point on the validated calibration curve for each analyte based on a signal-to-noise ratio minimum of 10:1 for the primary transition (quantitative transition), a precision of 20%, and an accuracy of 80–120% [37,38]. The accuracy for all analytes ranged from 91 to 108%. The LLOQ for each analyte were determined (along with their precision in %RSD) to be: 0.25 ng/mL for bima (3.5%), taf EA (2.3%), and 17-PTPF2␣ MA (3.1%); 0.5 ng/mL for bima IE (3.7%), bima SA (3.6%), clo IE (3.3%), lat (1.9%), trav (2.7%), and 16-PPF2␣ EA (2.4%); and 1 ng/mL for taf (1.9%), taf EE (3.4%), 17-PTPE2 SA (4.6%), 17-CF3 PTPF2␣ EA (1.8%), bima FA (3.0%), lat FA (1.1%), and clo FA-Na+ (2.8%). The limit of detection (LOD) was also established to be the lowest point on the validated calibration curve for each analyte based on a signal-to-noise ratio minimum of 3:1 for the secondary transition (identification transition). A weighting factor of 1/x2 , where x is the analyte concentration, was applied and all r values (correlation coefficient) were greater than 0.99. Both intraday (Table 2) and interday (Table 3) validations were performed. The standards were stable in the autosampler set at 4 ◦ C over the course of the stability studies (8 days). All stock and working solutions were stored at −20 ◦ C and were used over the course of 6 months. 3.4. Sample preparation The majority of the cosmetic products studied were water-based serums, and the remainder of the products were mascaras. Since the matrix of each product differed from the next, the QuEChERS liquid–liquid extraction (LLE) was implemented as the sample preparation technique. Although salt-out organic solvent (acetonitrile) extraction in QuEChERS has been applied to the analysis of pesticides, natural toxins, veterinary drugs, environmental pollutants, and other chemical contaminants in primarily food matrices, this universal approach has been applied for the first time, to the analysis prostaglandin analogs in cosmetic products. The modular approach of QuEChERS and small sample sizes allow for optimization to be easily achieved by the selection of the appropriate extraction solvent and rapid evaluation of a variety of clean-up solvents. For example, we were able to evaluate two different 2 mL dispersive solid-phase extraction (dSPE) tubes: one containing 50 mg Carbon X, 50 mg C18 , and 150 mg MgSO4 , and the other containing 50 mg C18 and 150 mg MgSO4 (United Science); were used as a clean-up step after the LLE, but were not implemented in the method due to a significant loss of the analyte into the sorbent (<50% recovery) and no increase in signal-to-noise ratio. Based on the recovery studies and method of standard addition results, the QuEChERS LLE was sufficient for sample preparation. 3.5. Recovery studies Recovery studies were carried out for all prostaglandin analogs. Recoveries for each analyte were calculated at three different levels: low (a mixture of 1 ng/mL each of 17-PTPF2␣ MA, bima, and taf EA: 2 ng/mL each of bima SA, 16-PPF2␣ EA, bima IE, lat, clo IE, and trav: 4 ng/mL each of bima FA, clo FA-Na+ , lat FA, 17-PTPE2 SA, 17-CF3 PTPF2␣ EA, taf EE, and taf), medium (a 5:10:20 ng/mL mixture), and high (a 25:50:100 ng/mL mixture) with respect to the calibrated range. A known amount of the analyte was spiked into a blank sample matrix and taken through the sample preparation process in triplicate. The recoveries of the analytes in the matrix were calculated as a percentage using the following equation: Recovery(%) =

Determined Amount × 100 Spiked Amount

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Fig. 2. Chromatograms of (a) and overlay of the primary transitions of all 16 prostaglandin analogs and (b) the primary and secondary transitions for bima IE observed for Product 5. Note: Identification of each analyte can be determined using the retention times in Table 1; three peaks (䊉) are observed due to impurities.

Table 2 Intraday validation. Run

1 2 3 4 5 6 Mean %RSD

Taf EA

Clo IE

17PF2␣ MA

Bima IE

r

Slope

Intercept

r

Slope

Intercept

r

Slope

Intercept

r

Slope

Intercept

0.99944 0.99487 0.99807 0.99910 0.99890 0.99885 0.99821 0.17

0.61045 0.62442 0.60216 0.58317 0.61235 0.60239 0.60582 2.27

0.01463 0.00947 0.01438 0.01888 0.02166 0.01627 0.01588

0.99937 0.99554 0.99906 0.99885 0.99921 0.99900 0.99833 0.16

0.26085 0.48884 0.50296 0.48797 0.49332 0.49485 0.49359 1.21

0.00431 –0.04398 0.00861 0.02729 0.03403 0.01465 0.00749

0.99865 0.99536 0.99911 0.99708 0.99790 0.99794 0.99767 0.13

0.44699 0.46336 0.44529 0.42847 0.44756 0.45698 0.44811 2.65

0.00132 0.02418 0.01642 0.05189 0.05660 0.02074 0.02853

0.99950 0.99640 0.99742 0.99885 0.99958 0.99755 0.99822 0.13

0.18117 0.18244 0.18289 0.18699 0.18991 0.18376 0.18453 1.78

0.02179 0.01419 0.00149 –0.00451 –0.00194 0.02559 0.00944

Note: Run 1 for Clo IE (red) was designated as an outlier and was not included in the mean and %RSD calculation.

Table 3 Interday validation. Day

1 2 3 4 5 8 Mean %RSD

Taf EA

Clo IE

17PF2␣ MA

Bima IE

r

Slope

Intercept

r

Slope

Intercept

r

Slope

Intercept

r

Slope

Intercept

0.99944 0.99895 0.99898 0.99869 0.99905 0.99968 0.99913 0.04

0.61045 0.60461 0.63254 0.64060 0.65384 0.59277 0.62247 3.77

0.01463 0.00343 0.00852 –0.00162 0.01022 0.01355 0.00812

0.99900 0.99875 0.99855 0.99933 0.99917 0.99966 0.99908 0.04

0.49485 0.46799 0.45683 0.52190 0.53057 0.65606 0.52137 13.8

0.01465 0.04199 0.00144 0.02439 0.01189 0.02057 0.01916

0.99865 0.99860 0.99924 0.99667 0.99897 0.99963 0.99863 0.10

0.44699 0.45280 0.46232 0.48660 0.47558 0.43511 0.45990 4.12

0.00132 0.03203 0.02701 –0.02478 0.03106 0.03516 0.01697

0.99950 0.99818 0.99544 0.99983 0.99767 0.99726 0.99798 0.16

0.18117 0.17526 0.17135 0.17774 0.17497 0.18643 0.17782 3.00

0.02179 0.03226 0.03366 –0.03673 0.00936 –0.03791 0.00374

Table 4 Recovery studies at the “low, medium, and high” levels of the calibration curves for all prostaglandin analogs (N = 3). Prostaglandin

% Recovery (%RSD) Low Level

% Recovery (%RSD) Medium Level

% Recovery (%RSD) High Level

17-PTPE2 SA Bima SA 16-PPF2␣ EA 17-PTPF2␣ MA Bima Bima FA Taf EA 17-CF3 PTPF2␣ EA Lat FA Clo FA-Na+ Taf EE Bima IE Lat Clo IE Taf Trav

100 (3.81) 105 (4.14) 108 (1.49) 102 (2.03) 99 (6.11) 99 (2.49) 95 (2.18) 87 (2.19) 99 (1.75) 107 (13.1) 97 (5.28) 75 (11.8) 106 (1.19) 104 (13.4) 109 (4.78) 115 (7.31)

98 (0.45) 100 (1.36) 103 (1.34) 100 (1.45) 102 (1.01) 98 (2.07) 97 (0.63) 95 (1.07) 99 (2.08) 99 (0.73) 99 (1.95) 99 (1.12) 99 (2.56) 128 (1.41) 102 (3.65) 104 (3.59)

97 (1.65) 99 (1.28) 103 (1.35) 100 (1.31) 102 (2.70) 99 (0.76) 97 (1.17) 97 (2.57) 101 (1.78) 95 (0.69) 100 (2.28) 97 (2.39) 100 (1.58) 120 (2.99) 98 (1.88) 100 (3.17)

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Table 6 Quantitation results for cosmetic products and prescription products containing prostaglandin analogs (N = 3).

Fig. 3. Plot of results for the method of standard additions for a sample of Product 12 + 17-PTPF2a MA.

where the determined amount was calculated using the corresponding calibration curve (Table 4). Recoveries for the majority of the analytes ranged from 95 to 109%. A more significant loss of signal was observed for the low-end recovery of 17-CF3 PTPF2␣ EA (87%) and bima IE (75%) while an enhancement was observed for the low-end recovery of trav (115%) and for the mid-to-high-end recovery of clo IE (128 and 120%, respectively). 3.6. Method of standard addition studies In order to evaluate if any matrix effects were occurring during the product survey the method of standard addition was conducted for three of the samples (Products 1, 6, and 12). A known amount of analyte was additionally added to a sample, taken through sample preparation, and quantified using the corresponding calibration curve. This process was carried out for the additions of 5, 15, and 35 ng/mL for each of the analytes. The ratio of analyte area-tointernal standard area was then plotted against the known amount of added analyte. A fitted line was applied, and the X-intercept was elucidated in order to obtain the absolute value of the “unknown” sample concentration (Fig. 3). The sample concentration calculated from the method of standard addition was then compared to the calculated concentration obtained without additional standard using the calibration curve (Table 5). A large difference in the two values would indicate a potentially significant matrix effect. The differences in the values calculated by the two methods for Products 1 and 12 were 6% and 1%, respectively, indicating no significant matrix effects. However, a difference of 18% was calculated for Product 6, indicating the possibility of a matrix effect. 3.7. Cosmetic product survey Thirty-one products were analyzed for containing one or more of 16 targeted prostaglandin analogs. Of the 31 products surveyed 13 tested positive (Table 6). Of the 16 targeted prostaglandin analogs only four (taf EA, bima IE, clo IE, 17-PTPF2␣ MA) were found in the commercial products studied, with clo IE being most prevalent followed by taf EA, bima IE, and 17-PTPF2␣ MA. The chromatogram in Fig. 2b shows the two transitions observed for bima IE during the survey of Product 5. The calculated concentrations for each were back-calculated to determine the amount of prostaglandin analog (␮g) per gram (g) of cosmetic product materials. Multiple samples of a few products with different batch numbers were also collected in order to confirm the consistency of Table 5 Method of standard addition results. Prostaglandin

Actual Conc. (ng/mL)

Calc. Conc. (ng/mL)

%Difference

Taf EA Clo IE 17-PTPF2␣ MA

5.65 6.38 6.66

5.98 5.22 6.62

6% 18% 1%

Product

Prostaglandin

Product 1 Product 2 Product 3 Product 4 Product 5 Product 6 Product 7 Product 8 Product 9 Product 10 Product 11 Product 12 Product 13 Prescription Product 1 Prescription Product 2 Prescription Product 3

Taf EA Taf EA Taf EA Bima IE Bima IE Clo IE Clo IE Clo IE Clo IE Clo IE Clo IE 17-PTPF2␣ MA 17-PTPF2␣ MA Trav Lat Bima

Calc. Conc. (%RSD) (␮g/g) 133 (11.3) 141 (8.04) 91.6 (10.6) 172 (4.90) 206 (5.98) 42.5 (1.40) 68.9 (6.78) 90.0 (3.89) 63.6 (6.44) 27.4 (6.68) 60.2 (5.09) 132 (2.07) 297 (1.19) 36.6 (8.05) 50.3 (7.63) 290 (4.15)

prostaglandin analog quantity within different batches. Three separate batches of Product 1 were analyzed for taf EA, and an average concentration of 141 ␮g/g (%RSD = 5.50) was determined. Two separate batches of Product 9 were analyzed for clo IE, and an average concentration of 70.4 (%RSD = 13.8) was determined. Although the prostaglandin analogs found in the cosmetic products were not the same prostaglandin analogs found in the prescription drugs the results were compared to available prescription products for glaucoma treatment (Prescription Product 1 and 2) and eyelash enhancement (Prescription Product 3) (Table 6). The concentration of prostaglandin analogs in the prescription products were labeled as follows: Prescription Product 1 (40 ␮g/mL travoprost), Prescription Product 2 (50 ␮g/mL latanoprost), and Prescription Product 3 (300 ␮g/mL bimatoprost). The density of each product was calculated by weighing 1 mL of the solution, in order to convert the known concentrations to ␮g/g, and was determined to be approximately 1 g/mL. The concentrations of the prescription products were all calculated to be within 9% of the labeled concentrations. Clearly, the concentrations of prostaglandin analogs in the surveyed cosmetic products are in a similar range when compared to available prescription products. 4. Conclusions In recent years, prostaglandin analogs have been used in cosmetic products for their eyelash and eyebrow lengthening, thickening, and darkening effects. Prior to this paper, no method had been developed for the quantitation of these chemicals in such products. To our knowledge, this is also the first account of the QuEChERS liquid–liquid extraction being utilized for the sample preparation of cosmetic products. This method has been developed, validated, and successfully applied to a number of cosmetic products for the quantitation of 16 prostaglandin analogs, and the results have been compared to known prostaglandin-containing prescription products. Acknowledgements James B. Wittenberg and Wanlong Zhou were supported by an appointment to the research participation program of the FDA administrated by the Oak Ridge Institute for Science and Education (ORISE). References [1] D. Jones, Enhanced eyelashes: prescription and over-the-counter options, Aesth. Plast. Surg. 35 (2011) 116–121.

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[2] P. Denis, D. Covert, A. Realini, Travoprost in the management of open-angle glaucoma and ocular hypertension, Clin. Ophtalmol. 1 (1) (2007) 11–24. [3] M.A. Teus, E. Arranz-Marquez, Prostaglandin F2␣ analogues in glaucoma management, Expert Rev. Ophthalmol. 3 (2) (2008) 203–209. [4] K.P.B Cracknell, I. Grierson, Prostaglandin analogues in the anterior eye: their pressure lowering action and side effects, Exp. Eye Res. 88 (2009) 786–791. [5] A.J. Patil, T.S. Vajaranant, D.P. Edward, Bimatoprost—a review, Expert Opin. Pharmacother. 10 (16) (2009) 2759–2768. [6] S. Fagien, Management of hypotrichosis of the eyelashes: focus on bimatoprost, Clin. Cosmet. Investig. Dermatol. 3 (2010) 39–48. [7] M. Digiuni, P. Fogagnolo, L. Rossetti, A review of the use of latanoprost for glaucoma since its launch, Expert Opin. Pharmacother. 13 (5) (2012) 723–745. [8] S.E. Easthope, C.M. Perry, Topical bimatoprost: a review of its use in open-angle glaucoma and ocular hypertension, Drugs Aging 19 (3) (2002) 231–248. [9] C.M. Perry, J.K. McGavin, C.R. Culy, T. Ibbotson, Latanoprost: an update of its use in glaucoma and ocular hypertension, Drugs Aging 20 (8) (2003) 597–630. [10] G. Holló, The side effects of the prostaglandin analogues, Expert Opin. Drug Saf. 6 (1) (2007) 45–52. [11] A. Alm, I. Grierson, M.B. Shields, Side effects associated with prostaglandin analog therapy, Serv. Ophthalmol. 53 (Suppl. 1) (2008) S93–S105. [12] K. Inoue, M. Shiokawa, R. Higa, M. Sugahara, T. Soga, M. Wakakura, G. Tomita, Adverse periocular reactions to five types of prostaglandin analogs, Eye 26 (2012) 1465–1472. [13] F.A. Fitzpatrick, High performance liquid chromatographic determination of prostaglandins F2␣ , E2 , and D2 from in vitro enzyme incubations, Anal. Chem. 48 (3) (1976) 499–502. [14] H. Salari, M. Yeung, S. Douglas, W. Morozowich, Detection of prostaglandins by high-performance liquid chromatography after conversion to p-(9anthroyloxy)phenacyl esters, Anal. Biochem. 165 (1987) 220–229. [15] B.-Y. Hsu, C.-Y. Tsao, T.-K. Chiou, P.-A. Hwang, D.-F. Hwang, HPLC determination for prostaglandins from seaweed Gracilaria gigas, Food Control 18 (6) (2006) 639–645. [16] S.S. Kumar, K. Natraj, A. Khan, B.K. Kumar, J.V. Rao, Development and validation of RP-HPLC method for estimation of bimatoprost in pharmaceutical dosage forms, J. Pharm. Res. 4 (10) (2011) 3733–3734. [17] V.L. McGuffin, R.N. Zare, Femtomole analysis of prostaglandin pharmaceuticals, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 8315–8319. [18] V.B. Ritov, D.E. Kelley, V.E. Kagan, Derivatization of F2 -isoprostanes with 1pyrenyldiazomethane and their subsequent determination by fluorescence high-performance liquid chromatography, Anal. Biochem. 311 (2002) 10–18. [19] J. Wübert, E. Reder, A. Kaser, P.C. Weber, R.L. Lorenz, Simultaneous solid phase extraction, derivatization, and gas chromatographic mass spectrometric quantification of thromboxane and prostacyclin metabolites, prostaglandins, and isoprostanes in urine, Anal. Chem. 69 (11) (1997) 2143–2146. [20] B.A. McCue, M.M. Cason, M.A. Curtis, R.D. Faulkner, D.C. Dahlin, Determination of travoprost and travoprost free acid in human plasma by electrospray HPLC/MS/MS, J. Pharm. Biomed. Anal. 28 (2002) 199–208. [21] P. Yang, E. Felix, T. Madden, S.M. Fischer, R.A. Newman, Quantitative highperformance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells, Anal. Biochem. 308 (2002) 168–177. [22] K. Nithipatikom, M.D. Laabs, M.A. Isbell, W.B. Campbell, Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells, J. Chromatogr. B 785 (2003) 135–145. [23] M. Koda, Y. Tsutsui, H. Niwa, S. Ito, D.F. Woodward, K. Watanabe, Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification

[24]

[25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

of bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS, Arch. Biochem. Biophys. 424 (2004) 128–136. P.J. Kingsley, C.A. Rouzer, S. Saleh, L.J. Marnett, Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry, Anal. Biochem. 343 (2005) 203–211. H. Cao, L. Xiao, G.Y. Park, X. Wang, A.C. Azim, J.W. Christman, R.B. van Breemen, An improved LC-MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids, Anal. Biochem. 372 (2008) 41–51. P.M. Krishna, B. Thirupathi Rao, M. Bujagendra Raju, C. Narasimha Rao, R. Kishore Kumar, P. Venkateswarlu, Determination of a novel impurity by LCMASS and chromatographic separation of bimatoprost, isomers and their impurities by UPLC, J. Pharm. Res. 4 (7) (2011) 2381–2383. A. Zammataro, C. Civiale, R. Saletti, S. Foti, Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body, J. Mass. Spectrom. 116 (2011) 8–117, 4. A. Furugen, H. Yamaguchi, N. Tanaka, H. Ito, K. Miyamori, A. Fujikawa, N. Takahashi, J. Ogura, M. Kobayashi, T. Yamada, N. Mano, K. Iseki, Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry, J. Chromatgr. B 879 (2011) 3378–3385. M. Enzler, S. Schipp, L.B. Nicolas, J. Dingemanse, C. Siethoff, Determination of 6-keto prostaglandin F1␣ and its metabolites in human plasma by LC-MS/MS, J. Chromatogr. B 901 (2012) 67–71. R. Takeshi, N. Adachi, Y. Yuzuki, L. Ito, Characterizatoin of prostaglandins by LC/APCI-MS, Bunskei Kagaku 43 (1994) 189–194. S.H. Lee, M.V. Williams, R.N. DuBois, I.A. Blair, Targeted lipidomics using electron capture atmospheric pressure chemical ionization mass spectrometry, Rapid Commun. Mass Spectrom. 17 (2003) 2168–2176. N. Cabaleiro, I. de la Calle, C. Bendicho, I. Lavilla, Current trends in liquid–liquid and solid–liquid extraction for cosmetic analysis: a review, Anal. Methods 5 (2013) 323–340. ˇ M. Anastassiades, S.J. Lehotay, D. Stajnbaher, F.J. Schneck, Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction: for the determination of pesticide residues in produce, J. AOAC Int. 86 (2) (2003) 412–431. S.J. Lehotay, K. Maˇstovská, S.J. Yun, Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes, J. AOAC Int. 88 (2) (2005) 630–638. S.C. Cunha, S.J. Lehotay, K. Maˇstovská, J.O. Fernandes, M. Beatriz, P.P. Oliveira, Evaluation of the QuEChERS sample preparation approach for the analysis of pesticide residues in olives, J. Sep. Sci. 30 (2007) 620–632. K. Zhang, J.W. Wong, P. Yang, K. Tech, A.L. KiBenedetto, N.S. Lee, D.G. Hayward, C.M. Makovi, A.J. Krynitsky, K. Banerjee, L. Jao, S. Dasgupta, M.S. Smoker, R. Simonds, A. Schreiber, Mulitresidue pesticide analysis of agricultural commodities using acetonitrile salt-out extraction, dispersive solid-phase sample clean-up, and high-performance liquid chromatography-tandem mass spectrometry, J. Agric. Food Chem. 59 (2011) 7636–7646. Y.-H. Park, K.-M. Joo, B.-Y. Woo, E.D. Son, S.Y. Byun, H.-J. Shin, K.-W. Lee, Y.-H. Park, K.-M. Lim, Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method, J. Pharm. Biomed. Anal. 61 (2012) 8–14. W. Zhou, P.G. Wang, O.A. Ogunsola, M.E.K. Kraeling, Rapid determination of hexapeptides by hydrophilic interaction LC-MS/MS for in vitro skin-penetration studies, Bioanalysis 5 (11) (2013) 1353–1362.