Zach C. Winfield

Here we highlight some of the opportunities associated with combining advanced sample preparation techniques with state-of-the-art chemical analysis techniques. This article considers the unique combination of selective pressurized liquid extraction (SPLE) with gas chromatography (GC) coupled with mass spectrometry (MS) and ultrahigh-pressure liquid chromatography coupled with tandem MS (UHPLC–MS-MS). We use this powerful combination to develop a novel analytical technique capable of measuring hormones and organic contaminants in whale earwax plugs. We explore the analytical challenges with such combinations and the advantages of focusing both on sample preparation as well as chemical analysis. 

	
	Sample preparation is a critical step in the analysis of organic compounds in solid and semisolid matrices and often involves multiple labor- and time-intensive steps, which in turn propagate uncertainty. Over the past decade, the automated extraction technique pressurized liquid extraction (PLE) has made significant advances to sample preparation as compared to classic techniques such as Soxhlet extraction. PLE is considered an exhaustive extraction technique suitable for a wide range of solid and semisolid matrices including tissues, soils, sediments, and particulate matter, as well as consumer products. To improve the extraction efficiency, PLE utilizes both high pressure (1500 psi) and high temperature (30–200 °C). Elevated temperatures serve to increase the solubility of the target analytes through accelerated extraction kinetics. Increasing the pressure helps ensure that the extraction solvent is below its boiling point. PLE typically uses between 20 and 100 mL of extraction solvent (depending on the size of the extraction cell) with extraction times of 15–30 min (depending on the static time and number of cycles). The reduction of extraction time and solvent are significant improvements compared to techniques like Soxhlet extraction and serve to increase analytical throughput. 
	
	PLE methods are optimized for specific analytes (typically with similar physical and chemical properties) through the assessment of solvent, temperature, extraction time, percent flush volume, number of cycles, and mass of matrix. During the extraction, target analytes and potential interference are extracted and collected together in a collection bottle. As a result, various cleanup strategies, such as packed chromatographic columns or gel-permeation chromatography, have been used to help remove potential interferences present in the extract. Complex matrices, such as biological tissues, often require multiple cleanup steps including the use of multiple packed chromatographic columns. These additional steps increase the time, training, space, and cost associated with the chemical analysis. Analytical trade-offs arise as matrix complexity and the number of target analytes increase.

	Recently, PLE techniques have integrated various cleanup strategies within the extraction cell and are often described as selective PLE (SPLE) or in-cell cleanup. SPLE is accomplished by layering commercially available adsorbents below the sample homogenate within the extraction cell. A wide range of commercially available adsorbents have been successfully incorporated within the extraction cell such as silica gel (including silica gel modified with sulfuric acid and potassium hydroxide), alumina, Florisil, carbon, diatomaceous earth, and C

. Subedi and colleagues (1) provided a detailed review of SPLE applications for organic contaminants.

	The incorporation of cleanup adsorbents within the extraction has been shown to eliminate some or all of the subsequent cleanup steps. The reduction of the number of analytical steps is advantageous on many levels including a reduction of training, space, and cost of consumables, as well as an increase in the overall analytical throughput and efficiency (that is, a reduction in analytical bottlenecks). These high-throughput SPLE methods also reduce or minimize the risk of sample contamination associated with sample preparation steps. Sample contamination is a major concern when measuring trace-level concentrations of organic contaminants in rare or irreplaceable samples.

	Marine mammals are often considered ecosystem sentinels because their survival is contingent on the health and functioning of marine ecosystems (2). Baleen whales (suborder 

) are unique sentinels because of their low trophic position and extensive geographic range, meaning that they are especially vulnerable to environmental perturbation. The bowhead whale (

), an Arctic species, is a sentinel in a habitat under rapid transition. As the extent and quality of sea ice decreases, this habitat is experiencing burgeoning impacts from oil and gas exploration, shipping activity, ocean noise, persistent contaminants, and commercial fishing-in addition to the ongoing ecological effects of climate change (3–5). Understanding and quantifying the exposure and effects of these stressors, such as persistent organic pollutants (POPs), on the bowhead whale is paramount to their conservation and management.

	Baleen whale earplugs are waxy structures that form annual or semiannual layers or lamina, and have classically been used in aging techniques (6,7). This waxy matrix consists of both a high lipid content (light layer) and keratinized epithelium cells (dark layer) (8). Recently, an SPLE technique was developed to measure a wide range of POPs in whale earwax (that is, cerumen [9]). One of the major advantages was the complete elimination of postextraction cleanup steps, which was accomplished by combining the necessary cleanup steps in the extraction step (9). In addition, enzyme-linked immunosorbent assay (ELISA) techniques have been developed to measure hormones in whale cerumen (10). It is important to note that each immunoassay kit is only capable of measuring a single hormone and that the analysis of multiple hormones would require significant sample mass, which may eliminate additional chemical measurements.

	POPs and hormone profiles were reconstructed for an individual whale with an estimated 6-month resolution, using the above mentioned methods to measure POPs and hormone concentrations in individual lamina (10). As a result, cerumen, and thereby earplugs, have the capability to record and archive chemical signals (both natural and anthropogenic). This technique is similar to chemical reconstruction techniques used in sediment or ice cores (11). This technique provides an unprecedented glimpse at a whale’s lifetime reproductive history, stress response, and contaminant exposure. This approach provides more accurate estimates of reproductive rate and age at sexual maturity than traditional methods and also yields baseline information regarding stress and contaminant exposure. One of the major issues associated with reconstructing chemical profiles using whale cerumen is the limited sample mass. Typically, the sample mass available for chemical analysis depends on the size of the earplug and, therefore, the age and species of the whale, but often ranges between 0.5 to 1.5 g/lamina. This limited sample mass reduces the overall number of chemicals or chemical classes that can be examined using a single earwax plug.

	The objective of this study is to expand on the SPLE method (9) to also include hormones, while preserving its ability to measure a wide range of POPs (Figure 1). Expanding the SPLE method would help maximize the number and type of analytes that can be reconstructed from a single whale earplug. Here, POPs are extracted using SPLE and extracts are analyzed using gas chromatography–mass spectrometry (GC–MS) (9). Hormones are eluted during a secondary extraction and those extracts are analyzed using ultrahigh-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS-MS). Liquid chromatography (LC) offers many advantages compared to traditional hormone analysis with immunoassay because smaller sample volumes can be used, multiple compounds can be measured from a single sample, and issues with cross reactivity are avoided (12). Hormones, such as testosterone, are typically more polar than the current list of POPs measured in whale earwax and offer a very unique set of analytical challenges. 


	
	This line of research has two main analytical challenges: First, developing an analytical technique capable of measuring part-per-billion concentrations of biologically relevant hormones in whale cerumen using UHPLC–MS-MS, and second, developing an SPLE technique that is capable of extracting contaminants as well as hormones from a single sample of whale cerumen without any additional cleanup steps.
	
	To optimize the SPLE technique, we need to maximize the extraction efficiency of the analytes from the matrix, in this case cerumen. To do so, we must simultaneously preserve the retention of our biological matrix compounds while extracting both organic contaminants and hormones. We believe we can extract both classes of compounds with the first extraction, but retain the hormones in the adsorbents along with the matrix itself. To do so, we must use adsorbents capable of retaining the lipid-rich matrix and hormones. Subsequently, the adsorbents should retain the bulk matrix while allowing the elution of the hormones with a secondary solvent extraction. A variety of solvents will be examined to produce the desired results.
	
	

	Unlabeled progesterone, estradiol, testosterone, cortisol, and labeled D

-cortisol with a purity of ≥98% were purchased from Cambridge Isotope Laboratories. All supplies were stored according to the manufacturer’s recommendations. Alumina (basic, activated, Brockmann I), and Florisil (60–100 mesh) were purchased from Sigma Aldrich. Silica gel (pore size 60 Å, 70–230 mesh) was purchased from BDH Chemicals. Concentrated formic acid and UHPLC-grade acetonitrile, ≥99.9% purity, were purchased from Fisher Scientific. For the UHPLC separations, a 100 mm × 2.1 mm, 1.7-µm 

 Acquity UPLC BEH C18 column and a 5 mm × 2.1 mm, 1.7-µm 

 UPLC BEH C18 VanGuard precolumn were purchased from Waters. Solvents and mobile phase were prepared using purified water (Thermo Barnstead Nanopure Diamond UV water purification, 18 Mâ¦).
	
	

SPLE for Organic Contaminants in Whale Earwax

	The SPLE technique capable of measuring contaminants (including pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers) in whale cerumen has been described previously (9). Briefly, aliquots of whale cerumen (∼0.25 g) were homogenized with anhydrous sodium sulfate and placed on precleaned adsorbents within the extraction cell (from top to bottom; 5 g of basic alumina, 15 g of silica gel, and 10 g of Florisil). All extractions were performed using an accelerated solvent extractor (ASE; ASE 350, Dionex [now part of Thermo Fisher Scientific]). Homogenates were extracted under the following extraction conditions with 1:1 dichloromethane–hexane, 100 °C, 1500 psi, 2 cycles (2 min each), and 150% rinse volume. Homogenates were spiked with isotopically labeled standards to correct for variability in analyte loss during sample preparation before SPLE. Extracts were spiked with a secondary set of isotopically labeled standards and concentrated to ~300 mL before GC–MS with electron ionization and GC–MS with electron capture negative ionization analysis.
	
	

	Hormone separation and analysis were performed using a Waters Acquity UPLC system and a Waters Xevo ESI/TQ-S electrospray ionization tandem MS system. Acetonitrile and water, with 0.1% formic acid, were selected along with a 100 mm × 2.1 mm, 1.7-µm 

 Acquity UPLC BEH C18 column with a 5-mm guard column with identical packing material and diameter. All samples were dried and reconstituted in 95:5 (v/v) water-acetonitrile with 0.1% formic acid following extraction. The column was initially equilibrated at 95% water (mobile-phase A) and 5% acetonitrile (mobile-phase B), for 30 min with a 0.5-mL/min flow rate. A quantitation method, using area, was established to monitor the following reactions (see Table I).

	Before analysis, the UHPLC system was flushed with 15 injections of water and 15 injections of acetonitrile at 0.7 mL/min with a 50% volume of 0.1% formic acid in water (mobile-phase A) and 0.1% formic acid in acetonitrile (mobile-phase B) to remove any waste from previous use. After it was cleaned, the column was loaded onto the instrument and conditioned at 95% A and 5% B for 30 min at 0.5 mL/min at 35 C. A target analyte calibration was performed with five points ranging from 2 ppb to 100 ppb using an isotopically labeled internal standard, then three blanks of 0.1% formic acid in water. The separation was programmed to begin at 5% B and increase to 40% B at 0.5 mL/min after 0.5 min and then gradually increase to 60% B for 4.5 min. The column was returned to initial conditions for 3.5 min before the separation was repeated. After the batch was complete, the column was flushed at initial conditions for 30 min and then flushed for 10 min in 95% B for storage.

SPLE Optimization for Hormones in Whale Earwax

	Typically, to optimize an SPLE method, a wide range of adsorbents, adsorbent masses, and combinations of adsorbents would be examined. However, because we are expanding on a previous SPLE method developed for the extraction and analysis of organic contaminants in whale earwax, the SPLE adsorbents have already been selected (adsorbents [basic alumina, silica gel, and Florisil]). At this juncture, we shift our focus to the hormones and acknowledge that hormones may be extracted from the whale earwax during the first extraction (extraction of contaminants using 1:1 dichloromethane–hexane) but retained on one or more of the adsorbents. In fact, our goal would be to extract both the contaminants and hormones during the first extraction step (dichloromethane–hexane) but retain the hormones for a secondary extraction on the adsorbents. In this ideal situation, the secondary extraction would elute the hormones from the adsorbents but preserve the retention of matrix compounds. The efficiency of extractions and adsorbents (retention of target analytes) were examined using target analyte percent recoveries and sample cleanliness (for example, effectiveness of adsorbents in retainment of interferences).

	The first step in optimizing the SPLE method for hormone analysis is to better understand the extraction of hormones from whale earwax using the prescribed SPLE adsorbents and solvent (dichloromethane–hexane; see below). This step is hindered such that specific hormones may be extracted from the earwax, but retained by one or more adsorbents. Hormones measured in the first extraction (dichloromethane–hexane) with high percent recoveries (>50%) would not be sufficiently retained in the extraction cell. Hormones with nondetects or very low recoveries (<10%) are assumed to be extracted, but retained by one or more adsorbent during the first extraction (dichloromethane–hexane). Past this point, a series of experiments must be designed to identify which hormones are retained by which adsorbents and which solvents or combinations of solvents are necessary to elute the hormones from the adsorbents.

	The extraction efficiency of multiple solvents and combinations of solvents were investigated as a potential second round extraction solvent (focusing on hormone elution). Dichloromethane, ethyl acetate, toluene, 1:1 dichloromethane–ethyl acetate, 2:1 ethyl acetate–dichloromethane, and cyclohexane were selected and examined based on literature information and polarity. Extractions were performed using a 100-mL cell containing 15 g of Florisil, 22.5 g of silica gel, and 7.5 g of basic alumina. All cells were conditioned (precleaned) using dichloromethane–hexane. Precleaning conditions were consistent throughout the experiments unless noted otherwise. Adsorbents were precleaned with a 1:1 dichloromethane–hexane at 100 C, 1500 psi, four cycles each with 2-min static times, a 60-s purge, and 50% flush volume. After precleaning, the cells were spiked with a solution of target analytes and left to equilibrate for 1 h at room temperature.

	The first extraction consisted of dichloromethane–hexane as previously described. The PLE conditions for each extraction were constant at 100 C, 1500 psi, two cycles each with 5-min static times, a 100-s purge, and 100% flush volume. 

	A following secondary extraction examined the extraction efficiency of different solvents (see above) and their ability to elute hormones retained on the adsorbents. In some specific cases, a third extraction was also examined and focused on very polar solvents including ethyl acetate and acetone. The second and third extractions used the PLE parameters described above and only varied by the solvent being examined. After the initial extraction of dichloromethane–hexane, all subsequent extractions were collected and analyzed separately. The samples were concentrated to ~1 mL using a Turbo Vap II evaporator (Biotage), then transferred to a GC vial and blown to dryness using compressed nitrogen with a fine blow-down peripheral. After drying, the sample was reconstituted in 475 mL of mobile phase-0.1% formic acid and 5% acetonitrile in water-then spiked with 25 mL of isotopically labeled internal standard before analysis using UHPLC–MS-MS.

	Target analyte affinity for individual adsorbents was examined with target analyte spike and recover experiments (

 = 1). Next, 10 g of each adsorbent were added to a 33-mL PLE cell. After conditioning (with dichloromethane–hexane; see above), each cell was spiked with a solution of target analytes as described above. Hormones were spiked on individual adsorbents packed into the extraction cell and were subsequently extracted using a range of extraction solvents. The extracts were then concentrated and reconstituted in the mobile phase as described above.

	Target analyte recoveries were also investigated with whale cerumen (0.25 g). A homogenate of multiple cerumen lamina created from a female bowhead whale (estimated at 65 years of age). A 0.25-g aliquot of the cerumen homogenate was homogenized with sodium sulfate (~10 g) and spiked in the extraction cell with target analyte. Cerumen samples, which provided a source of matrix interferences, were used to examine the matrix effects on 2:1 ethyl acetate–dichloromethane as the secondary extraction solvent. Extracts containing cerumen required a filter step to help remove precipitation, which appeared during blowdown. Filtering occurred after the extract was blown down to dryness and reconstituted in mobile phase (475 mL). A 13-mm Acrodisc CR filter with a 0.2-µm PTFE membrane (Pall Life Sciences) was precleaned with 500 mL of purified water. The samples were then drawn into a syringe and extruded through the filter into a clean GC vial.

	A third extraction was performed on two 66-mL cells, one with and one without earwax, both contained all three precleaned adsorbents. An extraction of ethyl acetate–dichloromethane (2:1) was performed on both cells. Once the extraction was complete the cells were allowed to cool to return to room temperature. The cell that did not contain any cerumen was extracted with ethyl acetate and subsequently with acetone. The cell cap of the cell containing wax was removed and the PLE extraction filter insertion tool was used to push the cellulose filter below the adsorbents upward. The layer of sodium sulfate and wax homogenate was scraped off carefully to leave behind the majority of basic alumina. After the homogenate was removed the sample was extracted with ethyl acetate followed by acetone.

	We began this novel method development with four hormones: cortisol, estradiol, progesterone, and testosterone. Hormone retention of each adsorbent was examined by placing 10 g of each adsorbent into a 33-mL extraction cell. The cells were conditioned with dichloromethane–hexane and then extracted with a range of different solvents. Analysis revealed that cortisol was well retained by all three adsorbents and preliminary results suggest that no solvent or combination was capable of eluting cortisol. Solvents and solvent combinations examined including 1:1 dichloromethane–ethyl acetate, 2:1 ethyl acetate–dichloromethane, ethyl acetate, toluene, ethyl acetate–toluene, and cyclohexane. The 2:1 ethyl acetate–dichloromethane solvent was selected over other solvents based on its ability to elute estradiol, progesterone, and testosterone. Toluene was capable of eluting select hormones, but required extensive blow-down time (3× as compared to 2:1 ethyl acetate–dichloromethane). Again, all solvent and solvent combinations were unable to elute cortisol off the adsorbents.

	Adsorbent experiments suggest that testosterone retention was dominated by silica gel. Spike and recovery experiments performed with all three adsorbents using 2:1 ethyl acetate–dichloromethane provided greater than 50% recovery. Percent recoveries were calculated by dividing the measured concentrations in the sample extract by expected concentrations and multiplying the quotient by 100%. A second experiment was performed in which cerumen (0.25 g cerumen homogenized with sodium sulfate) was loaded into a precleaned 66-mL cell and spiked with target analytes and left to equilibrate for 1 h. After it was extracted with 2:1 ethyl acetate–dichloromethane, the wax and sodium sulfate homogenate were extruded from the top of the cell and the three adsorbents were extracted with ethyl acetate. The ethyl acetate extraction provided a greater than 40% recovery of testosterone (Figure 2a).

	Adsorbent experiments suggest that estradiol retainment was dominated by Florisil. Spike and recovery experiments (without cerumen) performed with all three adsorbents using 2:1 ethyl acetate–dichloromethane provided greater than 80% recovery. Spike and recovery experiments (with cerumen) performed with all three adsorbents using 2:1 ethyl acetate–dichloromethane provided greater than 55% recovery. Again, a third extraction using ethyl acetate was performed after removing the sodium sulfate and wax homogenate. The ethyl acetate extraction provided a greater than 160% recovery of estradiol (Figure 2b). The higher estradiol recoveries were most likely caused by the native estradiol present in the female whale.

	Preliminary data suggest that hormones can be measured in whale earwax using an SPLE technique with a combination of extractions. Further parameters will be examined to improve the overall extraction efficiency, including the percent of flush volume, number of cycles, and static time. Future analysis will also include isotopically labeled target analytes to serve as surrogate standard. Surrogates will help correct for target analyte lose and variability in sample preparation.

	(1)   B. Subedi, L. Aguilar, E. Robinson, K. Hageman, E. Björklund, R. Sheesley, and S. Usenko, 

 119–132 (2015). 
	(2)   S.E. Moore, 

 534–540 (2008).
	(3)   C. Granier, U. Niemeier, J.H. Jungclaus, L. Emmons, P. Hess, J.-F. Lamarque, S. Walters, and G.P. Brasseur, 

(13), L13807 (2006).
	(4)   G.C. Hays, A.J. Richardson, and C. Robinson, T

 337–344 (2005).
	(5)   D.K. Perovich and J.A. Richter-Menge, 

 417–441 (2009).
	(6)   C.M. Gabriele, C. Lockyer, J.M. Straley, C.M. Jurasz, and H. Kato, 

 443–450 (2010).
	(7)   A. Jonsgard in 

 H.T. Andersen, Ed. (Academic Press, New York, 1969), pp. 1–30.
	(8)   P.E. Purves, 

 293–302 (1955).
	(9)   E.M. Robinson, S.J. Trumble, B. Subedi, R. Sanders, and S. Usenko, 

 14–20 (2013).
	(10) S.J. Trumble, E.M. Robinson, M. Berman-Kowalewski, C.W. Potter, and S. Usenko, 

 16922–16926 (2013).
	(11) S. Usenko, D.H. Landers, P.G. Appleby, and S.L. Simonich, 

 7235–7241 (2007).
	(12) C.J. Hogg, E.R. Vickers, and T.L. Rogers, 

J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 

 is with the Department of Chemistry and Biochemistry and the Department of Environmental Science at Baylor University in Waco, Texas. 

 is with the Department of Chemistry and Biochemistry at Baylor University. 

 is with the Department of Biology at Baylor University. 

is with the Department of Environmental Science at Baylor University. Direct correspondence to: 

Articles

This work addresses two challenges: developing a technique capable of measuring ppb levels of hormones, and developing an SPLE technique capable of extracting contaminants and hormones from a single sample without additional cleanup steps.

Zach C. Winfield

Articles

GC–MS and UHPLC–MS-MS Analysis of Organic Contaminants and Hormones in Whale Earwax Using Selective Pressurized Liquid Extraction