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Liquid chromatography–mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva
Journal of Chromatography B, 990 (2015) 158–163
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Liquid chromatography–mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva Ledicia Rey-Salgueiro, Elena Martínez-Carballo, Jesús Simal-Gándara ∗ Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Food Science and Technology Faculty, University of Vigo – Ourense Campus, E-32004 Ourense, Spain
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Article history: Received 21 January 2015 Received in revised form 19 March 2015 Accepted 24 March 2015 Available online 2 April 2015 Keywords: Solid-phase extraction Liquid chromatography/tandem mass spectrometry Pig breeds Saliva Stress hormones Corticosteroids
a b s t r a c t Biochemical response stressors results in an increase of adrenocortical activity. Before knowing the corticosteroid levels in saliva in a stressful situation, baselines salivary levels should be established. A method for simultaneous determination of five corticosteroids was developed, validated and applied to pig saliva at farms. The method employs solid-phase extraction (SPE) coupled with clean-up extraction step using silica cartridge in the same step followed by liquid chromatography/tandem mass spectrometry (LC–MS/MS), using electrospray ionization (ESI) in positive mode. The overall method quantification limits range from 0.050 to 0.30 g/L for the enrichment of 1.0 mL saliva samples and analyte recoveries are between 60 and 90% (RSD < 11%). Some factors studied were: pig sex, breeds, and time at farm. The analytical method clearly shows that CRL and CRS levels of, respectively, 3.0 and 4.0 g/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farm. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Swine welfare is of great importance in animal production systems and is usually evaluated using different indicators of stress [1]. Many existing methods used for assessing stress levels and consequently welfare tend to be invasive (viz. blood sampling), impractical (viz. collars for measuring heart rate) or time-consuming (viz. behavioural analysis) [2]. Given that abattoirs slaughter many pigs daily, there is a need for quick, reliable for assessing stress and at the same time allowing good decisions to be made at a relatively low cost [3]. Several studies showed that adaptation to saliva sampling of pigs was answered by no significant changes of heart rate and behaviour. In this way, cortisol (corticosteroid hormone) is one the most used biomarkers of stress in saliva, because of its concentration represent an adequate way to evaluate the hypothalamic–pituitary–adrenal (HPA) response to a stressor [4]. Inactive forms of cortisol represent cortisol’s reserve
Abbreviations: LC–MS, liquid chromatography–mass spectrometry; GC–MS, gas chromatography–mass spectrometry; ALD, aldosterone; CRS, corticosterone; CRL, cortisol; CRL-d4, cortisol-d4; DCRS, deoxycorticosterone; 11DCRL, 11deoxycortisol; 11DCRL-d5, 11-deoxycortisol-d5; C18, octadecyl-carbon; SPE, solid-phase extraction; PP, protein precipitation; LLE, liquid–liquid extraction. ∗ Corresponding author. Tel.: +34 988 387000; fax: +34 988 387001. E-mail address: [email protected] (J. Simal-Gándara). http://dx.doi.org/10.1016/j.jchromb.2015.03.021 1570-0232/© 2015 Elsevier B.V. All rights reserved.
pool in the time when more corticosteroids are needed (e.g. in stress response). There are disadvantages for the determination of corticosteroids in saliva, such as its low concentration (10% of that in blood), contamination problems with food or gastric fluid during sample collection and excessive handling thereof [4–6]. In order to solve all these drawbacks that may arise in the analysis of this marker, it is of paramount importance to have accurate and reproducible analytical methods for determining the levels of this kind of indicators in selected non-invasive samples. Several methods for determination of human salivary corticosteroids, mainly cortisol, have been reported but, to our knowledge, only radioimmuno and chemiluminescent assays for cortisol have been published in pig saliva [7,8]. However, the reliability of steroid immunoassays has been shown to be questionable because of the lack of specificity and matrix effects. Immunologic methods, especially direct assays, often overestimate steroid values [9,10]. Although immunoassays use liquid–liquid extraction to eliminate interfering compounds, these methods are still susceptible to interferences from other endogenous steroids [11]. Another limitation of immunoassays is the lack of an internal standard to monitor variable recovery of the extraction process [12]. In each case, several details about the practical procedure are missing for such complicate matrix. Alternatives to immunoassays are liquid and gas chromatography–mass spectrometry (LC–MS and GC–MS), which permit
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the highest specificity in steroid analysis. These techniques have the advantage that several steroid hormones can be measured simultaneously. However, GC–MS requires a time-consuming derivatization of the samples and therefore, GC step prior to MS prolongs the analytical run time considerably. Most current methods involve analysis by liquid chromatography coupled to tandem mass spectrometry [12,13]. In order to establish baseline levels of corticosteroids in pigs, which exhibit a circadian rhythm of plasma and saliva [14,15] with the highest levels in the morning, the aims of this paper are: (a) the development and characterization of an analytical method for controlling five corticosteroid stress hormones in pig saliva, and (b) the identification of baseline or non-stress levels of salivary corticosteroids in pigs according to their circadian rhythm.
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Table 1 LC–MS/MS conditions for the quantification of stress hormones and their precursors in pig saliva. Chromatographic conditions
LC–MS parameters
Injection volume Oven temperature Flow Solvent A Solvent B Solvent C Solvent programming
5 L 35 ◦ C 0.25 mL/min Water Methanol 10% HCOOH in watera 89% A + 10% B 59% A + 40% B in 4.0 min 29% A + 70% B in 4.0 min 19% A + 80% B in 4.0 min 4% A + 95% B in 0.2 min 4% A + 95% B for 3.8 min 89% A + 10% B in 0.2 min 89% A + 10% B for 4.0 min
2. Experimental 2.1. Chemicals and reagents Commercial hormones and precursors standards such as, aldosterone (ALD), corticosterone (CRS), cortisol (CRL), cortisold4 (CRL-d4), deoxycorticosterone (DCRS), 11-deoxycortisol (11DCRL) and 11-deoxycortisol-d5 (11DCRL-d5), were supplied by Sigma–Aldrich (Madrid, Spain). Octadecyl-carbon (C18) SPE C18, Sílica and Strata-X SPE cartridges were obtained from Phenomenex (Madrid, Spain) and Oasis HLB from Waters (Madrid, Spain). The solvents used (all purchased in HPLC-gradient grade), included methanol, acetone, diethyl ether, ethyl, acetate, n-hexane and water, as well as the reagent formic acid were from Sigma–Aldrich (Madrid, Spain). Stock standard solutions of individual compounds were prepared at 0.010 mg/mL (CRL-d4, 11DCRL-d5), 0.10 mg/mL (CRL, 11DCRL) and at 1.0 mg/mL (ALD, CRS, DCRS) in methanol and stored at −20 ◦ C. Multicompound working standard solutions were prepared in methanol by dilution of the stock solutions and stored at −20 ◦ C. 2.2. Animals and sampling Pietrain × (Landrace × Large White), Duroc × (Landrace × Large White) and L62 (crossed of several breeds) × (Landrace × Large White) female and male pigs (barrow for Duroc and intact male pigs for the remaining breeds) were used in this study, weighing approximately 88 kg and 15 weeks of age. The procedure was carried out in a commercial farm where all relevant farmed animal welfare requirements are met. Pigs were housed in groups of 10–12 animals (stocking density of 1.0 m2 /pig; 88 kg/m2 ) and male and female pigs were kept in different stables. Saliva samples were taken at 8:00–8:30, 10:00–10:30, 12:00–12:30 and at 14:00–14:30 from animals housed in four pens (two replicates per sex) from three farms, one for each breed. A total of 48 saliva samples were collected. Saliva was collected by suspending twisted 100% cotton ropes (not chemically treated) into each pen at approximately 40 cm above from the floor for 30 min [16]. Pigs are naturally attracted to the rope and deposit oral fluids as they chew it. A period of time of 30 min was enough for 75% of the animals contacting with the rope in a stable of 25–30 animals [17]. After the exposure period, saliva samples were extracted from the rope by wringing the wet end into a plastic bag and clipping a bottom corner of the bag to drain the fluid into a tube. Then, samples were centrifuged (590 g/10 min) and were stored at −80 ◦ C until assayed.
Detection conditions Compound
Transition reactions (m/z)
CRL CRL-d4 11DCRL 11DCRL-d5 CRS DCRS ALD
363/121 367/121 347/109 352/112 347/121 373/109 361/343
a During the gradient LC separation the concentration of formic acid was constant at 0.1% (v/v).
contains a quaternary pump, auto sampler, degasser and column compartment. Chromatographic separations were performed with ˚ LC Column 100 × 2.1 mm) (Phenoa KinetexTM 2.6 m C18 (100 A, menex, Torrance, CA, USA). Chromatographic conditions are given in Table 1. A TSQ Quantum Discovery triple stage quadrupole mass spectrometer equipped to an electrospray interface from Thermo Fisher Scientific was used as detector. MS/MS analysis was performed using argon as the collision gas and nitrogen as the nebulizer gas. Quantification was performed using selected reaction monitoring (SRM) in positive mode of precursor > product ion transitions (see Table 1). Capillary voltage was set to 4.0 kV. Capillary temperature of 350 ◦ C, sheath gas and auxiliary gas pressure of 40 and 5.0 psi, Tube lens of 120 V and collision energy of 25 eV (1.5 mTorr collision cell pressure) were selected. 2.4. Extraction and purification procedure The extraction and purification procedure of the target stress hormones in pig saliva was performed by SPE with C18 and silica cartridges in series. Saliva samples (1.0 mL) were firstly passed through a C18 cartridge, which was previously activated with 3.0 mL of methanol and 1.5 mL of water. The cartridge was then washed with 0.25 mL of water, followed by 0.50 mL of water:acetone (4:1) and 0.25 mL of hexane. Then, a cartridge of silica is set in connection with the C18, which was activated beforehand with 2.0 mL of hexane followed by 1.0 mL of ethyl acetate. The hormones and precursors were then eluted from the C18-silica cartridges by passing through 6.0 mL of ethyl acetate:ethyl ether (1:1). The eluate was then evaporated to dryness, dissolved in 100 L of methanol, centrifuged at 850 g for 10 s and the supernatant was analysed by LC–MS/MS. 2.5. Analysis of variance (ANOVA) and multiple comparison test
2.3. Chromatography and mass spectrometry The liquid chromatographic system used was a Dionex Ultimate 3000 HPLC system Thermo Scientific (Madrid, Spain), which
The statistical analyses were performed with the statistical software package Statgraphics Plus v. 5.1 (Manugistics, Rockville, MD, USA). Significant differences in CRL and CRS levels amongst the
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different pig groups (separated by breed: Duroc, Pietrain and L62; and time: 0, 2.0, 4.0 and 6.0 h) were evaluated by one-way and two-way analysis of variance (ANOVA) at the 95% confidence level. A Fisher’s least significant difference (LSD) test, at a 95% confidence level, was also used to detect interactions amongst the variables. Pearson correlations among the levels of the different stress hormones in pig saliva were also performed. 3. Results and discussion One of the objectives of this work was to develop and optimize an analytical method for determining five corticosteroid hormones in pig saliva, which will serve as a tool for understanding the baseline levels in pigs at farms. Pig saliva is a very complex matrix and differs strongly with the human saliva. Human saliva is collected in special conditions (fasting conditions, 2.0 h after eating or drinking or rinse their mouths with cold water at least). These conditions could not be used for animals such as pigs in commercials farms and pig saliva samples contain feed or other interferences. Therefore a more exhaustive clean-up procedure prior to detection was required. Pre-treatment of the sample, method characterization and possible matrix effects were evaluated using spiked saliva samples (15 g/L for ALD and 5.0 for g/L for CRL, CRS, 11DCRL and DCRS). Once the analytical methodology was validated, it was applied to saliva samples for setting baseline non-stress salivary corticosteroids levels at pig farms, which is the second objective. 3.1. Optimisation of liquid chromatographic separation Chromatographic conditions were optimised for the simultaneous determination of CRL, CRS, 11DCRL, DCRS and ALD. To our knowledge, some studies about the determination of several hormones in different biological samples have been published.
Nevertheless none of them selected the target analytes [18,19]. After assaying different mobile phase systems and gradients, the mobile phase water/methanol/10% formic acid was selected (Table 1). Good chromatography is important for the analysis of steroids to ensure the adequate separation of isobaric compounds i.e. with the same mass to charge ratio (11DCRL and CRS), which all share the same nominal molecular weight and are indistinguishable in the mass spectrometer. For this reason different HPLC ˚ LC Colcolumns including a KinetexTM 2.6 m Biphenyl (100 A, ˚ LC Column 100 × 2.1 mm) umn 50 × 2.1 mm)TM , 2.6 m C18 (100 A, ˚ LC Column 100 × 4.6 mm, Ea) from and Luna® 3 m C18(2) (100 A, ˚ LC Phenomenex were tested. Only with the 2.6 m C18 (100 A, Column 100 × 2.1 mm) LC column the separation between the target isobaric compounds (11DCRL and CRS) was completely in only 20 min. The greater efficiency of the selected column increases peak resolution and improves sensitivity by giving sharp peaks with better signal to noise characteristics. The optimised conditions described above allowed the elution of hormones and their precursors within 20 min (Fig. 1). 3.2. Sample treatment The most used saliva sample treatment strategies comprise protein precipitation (PP), solid-phase extraction (SPE) and liquid–liquid extraction (LLE) [20]. Therefore, the first step of our research was to test these procedures. 3.2.1. Precipitation of salivary proteins Precipitation of proteins is a pre-treatment step for determination of glucocorticoids as CRL in biological fluids by other authors [21,22]. In this study, different protein precipitants were tested to obtain a cleaner sample and to reduce interfering substances. In this way acetonitrile acidified with 0.50% (v/v) acetic and 10% (v/v)
Fig. 1. LC–MS/MS chromatograms: A) standard chromatogram at 50 g/L (except DCRS at 25, and ALD at 100 g/L), B) saliva extract, and C) SRM chromatogram for 1: ALD, 2: CRL, 3: CRS, 4: 11DCRL, 5: DCR.
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trichloroacetic acid in cold acetone were evaluated. The method was carried out according to Jönsson et al. [22], both precipitants were added at 1.0 mL of spiked saliva sample and after 10 min at room temperature were centrifuged at 690 × g for 15 min. Then, the supernatant was evaporated in a nitrogen flow and dissolved in 100 L methanol for LC–MS/MS analysis. In this way, the final extract contained numerous smaller particles from the complex saliva sample, which could not be separated by centrifugation or filtering (on nylon and PTFE filter). The extract was not able to LC–MS/MS analysis due to it higher suspension viscosity of the final extract and therefore, only the step of protein precipitation was not enough or suitable for cleaning the extract from pig samples. 3.2.2. Extraction Because of this drawback obtained with the protein precipitation, two extraction procedures were assayed: 3.2.2.1. Liquid–liquid extraction. Recent studies showed that LLE is an effective sample preparation technique for analysis of steroids. Jensen et al. [23] and Matsui et al. [24] developed a method based on LLE followed by evaporation to dryness and reconstitution in an organic solvent for the determination of salivary CRL. According to these authors, LLE was carried out by adding 2.0 mL ethyl acetate to 1.0 mL of a spiked saliva sample. The resulting mixture was subsequently shaken for 45 min on a horizontal shaker. The samples were centrifuged for 5 min at 3500 × g and then the supernatant was evaporated to dryness under nitrogen. Finally, the residue was redissolved in 100 L methanol but a very complex extract was again obtained. 3.2.2.2. SPE extraction. SPE was frequently developed for the extraction of glucocorticoids from biological fluids. De Palo et al. [25], managed to solve the quantitative extraction of human saliva samples for measurement of CRL and cortisone using a C18. AbuRuz et al. [26] and Oledzka et al. [27] used HLB cartridges for the extraction of prednisolone, CRS and CRL from plasma and urine samples, observing quantitative recoveries. Han et al. [28] described an extraction procedure for 6-hydroxycortisol and free CRL based on Strata-X cartridges and provided recoveries of more than 93% for both analytes. In the present study, Strata-C18, Strata-X and HLB cartridges were tested. Based on the literature, several organic solvents were evaluated for conditioning, clean up and elution of cartridges. Based on the previous studies, the cartridges were first conditioned by washing with methanol and water, and the saliva sample was then loaded onto the cartridge. After a further rinse with water, followed by water:acetone (4:1) and hexane, the analytes were eluted by passing through ethyl acetate:ethyl ether (1:1). Finally, the eluate was evaporated to dryness, dissolved in 100 L of methanol. Volumes of the solvents were optimized in order to obtain the highest recoveries. Although SPE extracts were more suitable to detect by LC–MS/MS than the obtained with PPT and LLE, they still showed impurities. For this reason in the present work an additional cleanup step was added. 3.2.3. Clean up Strata-Silica cartridge was connected in series with each cartridge evaluated above (C18, Strata-X and HLB). Therefore only activation step for silica cartridge was optimized. In this way, after wash step, according to the procedure described above, the silica cartridge was inserted (previously activated with hexane followed by ethyl acetate) and the analytes were then eluted from the two cartridges connected in series. In this way for the first time, quantitative recoveries were obtained (Table 2a). The best results were achieved for C18 and silica cartridges in series. Therefore, these
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cartridges were selected to the extraction and purification of stress hormones and precursors under study from pig saliva. 3.3. Performance of the analytical method Molecules originating from the sample matrix that coelute with the compound(s) of interest can interfere with the ionization process in the mass spectrometer, causing ionization suppression or enhancement [29,30] the so-called matrix effect. In order to compensate for the alteration in signal and method fluctuations, some internal standards with similar molecular properties as the analytes under study (CRL-d4 and 11CRL-d5) were tested. The post-extraction addition technique is usually used to determine the degree of matrix effects on an LC–ESI–MS/MS method. The difference in response between the post-extraction sample and the pure solution divided by the pure solution response determines the degree of matrix effect occurring to the analyte in question under chromatographic conditions [31]. In the present study, the matrix effect was determined as around −30% for compounds under study and CRL-d4, representing a loss of 30% of the target analytes signal (ion suppression) due to alterations in ionization efficiency. Nevertheless, 11CRL-d5 was suitable internal standard only for 11CRL and different degrees of matrix effects were found for the other target analytes. CRL-d4 was then selected as internal standard. Method performance was assessed by evaluating several quality parameters of the method such as recovery values, repeatability, reproducibility, linearity and limits of detection and quantification. For this purpose, saliva samples were previously fortified with the target analytes at different levels and treated following the experimental conditions described in Section 2.3. For the assessment of the linearity of the method, a saliva sample was fortified with the target analytes at different levels by triplicate (n = 6, ranging from 0.40 to 25 g/L) and subjected to the optimized protocol. About 10 L of 500 g/L of internal standard was added to the fortified saliva samples and internal calibration was carried out. To statistically validate the regression analysis, the linearity was verified by the Mandel fitting test (P = 99%) [32]. Acceptable linearity was obtained for each compound evaluated with a correlation coefficient >0.9920 (Table 2A). To confirm that the matrix effects are corrected, recoveries of each compound were evaluated from the ratio between the slope of the calibration curve of the spiked samples and the slope of the calibration line corresponding to the hormones standards. The concentrations considered for both calibration lines ranged from 0.40 to 25 g/L (n = 6 in triplicate). The recoveries obtained ranged from 74% to 90% with the exception of ALD (60%) (Table 2B). Repeatability (within-day precision) and reproducibility (between-day precision) studies were calculated by analysing a saliva sample. In order to determine the repeatability of the method, replicate analysis were carried out on the same day (n = 5) and the reproducibility was tested over 3 consecutive days by performing five repeated analysis each day (n = 15), respectively. As can be seen in Table 2B, the relative standard deviations (RSD%) for both, repeatability and reproducibility, was lower than 11%. These RSD values testify to the excellent ruggedness of the method. Limits of detection (LOD) and quantification (LOQ) were calculated in an unfortified saliva sample following the signal-to-noise criteria (S/N = 3 and S/N = 10 for LODs and LOQs, respectively) [33]. The estimated LODs ranged from 0.020 to 0.10 g/L, whereas LOQs ranged from 0.050 to 0.31 g/L for the tested analytes (Table 2B). 3.4. Determination of stress hormones and precursors in pig saliva Once the analytical methodology was validated, it was applied to saliva samples collected in different pig farms. Forty-eight saliva
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Table 2 Recoveries comparing different SPE cartridges (% ± SD) in (A), and performance of the optimized method with the C18 cartridge for the determination of stress hormones and their precursors in pig saliva (B). (A) Compound
C18
Strata-X
HLB
CRL 11DCRL CRS DCRS ALD
74 ± 5.3 86 ± 8.1 90 ± 6.9 59 ± 4.3 74 ± 8.3
85 ± 8.4 <50 59 ± 7.3 <50 <50
55 ± 5.8 <50 66 ± 6.2 <50 <50
(B) Compound
Recoverya (%R)
Repeatabilitya (%RSD)
Reproducibilityb (%RSD)
Linear dynamic rangec (g/L)
Determination coefficient (r2 )
LOD (g/L)
LOQ (g/L)
CRL 11DCRL CRS DCRS ALD
74 86 90 60 74
5.3 6.9 8.1 4.3 8.3
7.3 7.5 10.7 6.6 9.4
0.40–10.0 0.5–10.0 0.75–10.0 0.5–10.0 2.0–25.0
0.9987 0.9991 09994 0.9920 09954
0.020 0.020 0.030 0020 0.100
0.050 0.050 0.080 0.050 0.30
a b c
n = 5. n = 15. n = 6.
samples (three breeds (Duroc, L62 or Pietrain) × two sex (male or female) × four sampling times (0, 2.0, 4.0 and 6.0 h) in duplicate) were analysed. A chromatogram of a saliva sample is shown in Fig. 1B. Fig. 2 shows the results for the selected saliva samples. As can be seen, only CRL and CRS were detected in the sampling saliva samples of pigs at farms. No significant differences in hormone levels were found between sexes (two integrated samples from 12 pigs were compared per sex). Data were then pooled for comparison at 0, 2.0, 4.0 and 6.0 h (four integrated samples per time) and no significant differences were found for any of the pig breeds although the concentrations of CRL had a clear gradient for breeds: Duroc > L62 = Pietrain. It was also found that significantly higher basal plasma CRL levels were detected in Erhualian breed [34,35]. This breed is well-known for their high fertility and superior meat quality with high intramuscular fat content and these characteristics are common in Duroc breed. The breed differences found in basal CRL levels were suggested to reflect the fundamental
differences in adrenocortical function [36]. As it was also previously documented, the baseline salivary CRL concentrations in pigs differ with housing conditions [37,38]. In the present study, CRL concentrations were within the same range as has been found previously for pigs housed under similar conditions (sampling time and under intensive conditions in a barren and restricted environment) [37,38]. Similar non-stress levels of CRL were found also by Geverink et al. [39] and Schönreiter and Zanella [40] in their studies about responses to different stressors. No differences were also found for CRS for among the breeds, but their levels trend to be higher than those of CRL (Fig. 2). Usually, the levels of CRS were clearly higher in the central hours of the morning as compared to those of CRL. Pearson correlations among the levels of the different stress hormones in pig saliva were found to be significant for CRL and CRS (n = 48, r = 0.524, P < 0.0001), which is a clear fact that they have similar responses to stress, indicating that both hormones could be stress indicators, and therefore CRS could be also used as biomarker of non-stress in saliva. Based on the results obtained as well as the results obtained by other authors, CRL and CRS levels of, respectively, 3.0 and 4.0 g/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farms. Stressors increasing their levels could be of many different origins, e.g. ecological (acute environmental changes, nutrition or shelters absence, temperature variations, etc.), socio-biological (unstable social hierarchy), health (fever, infection, injury, surgery, etc.), transport and many others. All these stressors trigger stress response – a complex of physiological, endocrine, metabolic and behaviour reactions protecting organism prior to the injurious effect of stressors (emergency life-history) [4].
4. Conclusions
Fig. 2. CRL and CRS levels compared regarding breed (Duroc, D; L, L62; and Pietrain, P). No differences at 95% probability were found for both pig sexes (male and female), but also for time between 8:00 and 14:30 h (time 0 = 8:00–8:30, time 2 = 10:00–10:30, time 4 = 12:00–12:30 and time 6 = 14:00–14:30). For these reasons, data were pooled.
An analytical method for the determination of five corticosteroids (DCRS, CRS, ALD, 11DCRL and CRL), as porcine stress biomarkers in saliva was developed and optimized. Quantitative recoveries were obtained using SPE with C18 and silica cartridges at the same time in order to perform extraction and clean up in the same step. LC–MS/MS with ESI in positive mode was used to detect the target corticosteroids. This method was applied to saliva samples from three breeds of pigs (Duroc, L62 and Pietrain) at farms in order to establish basal levels of the target corticosteroids. Since only CRL and CRS were detected in the selected samples and a significant correlation was detected between their levels, we could
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hypothesize that CRL and CRS could be used both as biomarkers of non-stress in saliva of pigs. CRL and CRS levels of, respectively, 3.0 and 4.0 g/L in saliva could be indicative of maxima non-stress levels in different pig breeds at farm. Acknowledgements This work was supported by the project INNOSABOR (ITC20133017) which is supported by the FEDER-INNTERCONECTA program from the CDTI (Centre for Industrial Technological Development). The authors are grateful to Ruth Lois, Miguel Sarria and Borja Casales for their help with the planning of the experimental set-up at farms. References [1] V. Caporale, B. Alessandrini, P. Dalla Villa, S. Del Papa, Global perspectives on animal welfare: Europe subscription status, OIE Rev. Scientif. Techniq. 24 (2005) 567–577. [2] J. Lane, J. Flint, C. Danks, The development of a rapid diagnostic test for cortisol in the saliva of pigs, Intern. J. Appl. Res. Vet. Med. 7 (2002) 4. [3] M.L. Seshoka, A.T. Kanengoni, F.K. Siebrits, K.H. Erlwanger, The novel use of “point of care” devices to evaluate transport duration on selected pork quality parameters, S. Afr. J. Anim. Sci. 43 (2013) S48–S53. [4] E. Möstl, R. Palme, Hormones as indicators of stress, Domest. Anim. Endocrin. 23 (2002) 67–74. [5] N. Tadich, C. Gallo, H. Bustamante, M. Schwerter, G. Van Schaik, Effects of transport and lairage time on some blood constituents of Friesian-cross steers in Chile, Livest. Prod. Sci. 93 (2005) 223–233. [6] C. Tsigos, G.P. Chrousos, Hypothalamic-pituitary-adrenal axis neuroendocrine factors and stress, J. Psychosom. Res. 53 (2002) 865–871. [7] N.J. Cook, A.L. Schaefer, P. Lepage, S. Morgan Jones, Salivary vs serum cortisol for the assessment of adrenal activity in swine, Can. J. Anim. Sci. 76 (1996) 329–335. [8] D. Escribano, M. Fuentes-Rubio, J.J. Cerón, Validation of an automated chemiluminescent immunoassay for salivary cortisol measurements in pigs, J. Vet. Diagn. Invest. 24 (2012) 918–923. [9] D.A. Herold, R.L. Fitzgerald, Immunoassays for testosterone in women: better than a guess? Clin. Chem. 49 (2003) 1250–1251. [10] F.Z. Stanczyk, M.M. Cho, D.B. Endres, J.L. Morrison, S. Patel, R.J. Paulson, Limitations of direct estradiol and testosterone immunoassay kits, Steroids 68 (2003) 1173–1178. [11] S.A. Wudy, U.A. Wachter, J. Homoki, W.M. Teller, 17-Alphahydroxyprogesterone 4-androstenedione and testosterone profiled by routine stable isotope dilution/gas chromatography–mass spectrometry in plasma of children, Pediatr. Res. 38 (1995) 76–80. [12] M. Rauh, M. Gröschl, W. Rascher, H.G. Dörr, Automated fast and sensitive quantification of 17␣-hydroxy-progesterone androstenedione and testosterone by tandem mass spectrometry with on-line extraction, Steroids 71 (2006) 450–458. [13] R. Miller, F. Plessow, M. Rauh, M. Gröschl, C. Kirschbaum, Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry, Psychoneuroendocrinology 38 (2013) 50–57. [14] E.D. Ekkel, S.J. Dieleman, W.G.P. Schouten, A. Portela, G. Cornélissen, M.J.M. Tielen, F. Halberg, The circadian rhythm of cortisol in the saliva of young pigs, Physiol. Behav. 60 (1996) 985–989. [15] M.A.W. Ruis, J.H.A. Te Brake, B. Engel, E.D. Ekkel, W.G. Buist, H.J. Blokhuis, J.M. Koolhaas, The circadian rhythm of salivary cortisol in growing pigs: effects of age gender and stress, Physiol. Behav. 62 (1997) 623–630. [16] J.R. Prickett, W. Kim, R. Simer, K.J. Yoon, J. Zimmerman, Oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections, J. Swine Health Prod. 16 (2008) 86–91. [17] S.E. Detmer, D.P. Patnayak, Y. Jiang, M.R. Gramer, S.M. Goyal, Detection of influenza a virus in porcine oral fluid samples, J. Vet. Diagn. Invest. 23 (2011) 241–247. [18] T. Guo, R.L. Taylor, R.J. Singh, S.J. Soldin, Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry, Clin. Chim. Acta 372 (2006) 76–82.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Liquid chromatography–mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva Ledicia Rey-Salgueiro, Elena Martínez-Carballo, Jesús Simal-Gándara ∗ Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Food Science and Technology Faculty, University of Vigo – Ourense Campus, E-32004 Ourense, Spain
a r t i c l e
i n f o
Article history: Received 21 January 2015 Received in revised form 19 March 2015 Accepted 24 March 2015 Available online 2 April 2015 Keywords: Solid-phase extraction Liquid chromatography/tandem mass spectrometry Pig breeds Saliva Stress hormones Corticosteroids
a b s t r a c t Biochemical response stressors results in an increase of adrenocortical activity. Before knowing the corticosteroid levels in saliva in a stressful situation, baselines salivary levels should be established. A method for simultaneous determination of five corticosteroids was developed, validated and applied to pig saliva at farms. The method employs solid-phase extraction (SPE) coupled with clean-up extraction step using silica cartridge in the same step followed by liquid chromatography/tandem mass spectrometry (LC–MS/MS), using electrospray ionization (ESI) in positive mode. The overall method quantification limits range from 0.050 to 0.30 g/L for the enrichment of 1.0 mL saliva samples and analyte recoveries are between 60 and 90% (RSD < 11%). Some factors studied were: pig sex, breeds, and time at farm. The analytical method clearly shows that CRL and CRS levels of, respectively, 3.0 and 4.0 g/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farm. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Swine welfare is of great importance in animal production systems and is usually evaluated using different indicators of stress [1]. Many existing methods used for assessing stress levels and consequently welfare tend to be invasive (viz. blood sampling), impractical (viz. collars for measuring heart rate) or time-consuming (viz. behavioural analysis) [2]. Given that abattoirs slaughter many pigs daily, there is a need for quick, reliable for assessing stress and at the same time allowing good decisions to be made at a relatively low cost [3]. Several studies showed that adaptation to saliva sampling of pigs was answered by no significant changes of heart rate and behaviour. In this way, cortisol (corticosteroid hormone) is one the most used biomarkers of stress in saliva, because of its concentration represent an adequate way to evaluate the hypothalamic–pituitary–adrenal (HPA) response to a stressor [4]. Inactive forms of cortisol represent cortisol’s reserve
Abbreviations: LC–MS, liquid chromatography–mass spectrometry; GC–MS, gas chromatography–mass spectrometry; ALD, aldosterone; CRS, corticosterone; CRL, cortisol; CRL-d4, cortisol-d4; DCRS, deoxycorticosterone; 11DCRL, 11deoxycortisol; 11DCRL-d5, 11-deoxycortisol-d5; C18, octadecyl-carbon; SPE, solid-phase extraction; PP, protein precipitation; LLE, liquid–liquid extraction. ∗ Corresponding author. Tel.: +34 988 387000; fax: +34 988 387001. E-mail address: [email protected] (J. Simal-Gándara). http://dx.doi.org/10.1016/j.jchromb.2015.03.021 1570-0232/© 2015 Elsevier B.V. All rights reserved.
pool in the time when more corticosteroids are needed (e.g. in stress response). There are disadvantages for the determination of corticosteroids in saliva, such as its low concentration (10% of that in blood), contamination problems with food or gastric fluid during sample collection and excessive handling thereof [4–6]. In order to solve all these drawbacks that may arise in the analysis of this marker, it is of paramount importance to have accurate and reproducible analytical methods for determining the levels of this kind of indicators in selected non-invasive samples. Several methods for determination of human salivary corticosteroids, mainly cortisol, have been reported but, to our knowledge, only radioimmuno and chemiluminescent assays for cortisol have been published in pig saliva [7,8]. However, the reliability of steroid immunoassays has been shown to be questionable because of the lack of specificity and matrix effects. Immunologic methods, especially direct assays, often overestimate steroid values [9,10]. Although immunoassays use liquid–liquid extraction to eliminate interfering compounds, these methods are still susceptible to interferences from other endogenous steroids [11]. Another limitation of immunoassays is the lack of an internal standard to monitor variable recovery of the extraction process [12]. In each case, several details about the practical procedure are missing for such complicate matrix. Alternatives to immunoassays are liquid and gas chromatography–mass spectrometry (LC–MS and GC–MS), which permit
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the highest specificity in steroid analysis. These techniques have the advantage that several steroid hormones can be measured simultaneously. However, GC–MS requires a time-consuming derivatization of the samples and therefore, GC step prior to MS prolongs the analytical run time considerably. Most current methods involve analysis by liquid chromatography coupled to tandem mass spectrometry [12,13]. In order to establish baseline levels of corticosteroids in pigs, which exhibit a circadian rhythm of plasma and saliva [14,15] with the highest levels in the morning, the aims of this paper are: (a) the development and characterization of an analytical method for controlling five corticosteroid stress hormones in pig saliva, and (b) the identification of baseline or non-stress levels of salivary corticosteroids in pigs according to their circadian rhythm.
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Table 1 LC–MS/MS conditions for the quantification of stress hormones and their precursors in pig saliva. Chromatographic conditions
LC–MS parameters
Injection volume Oven temperature Flow Solvent A Solvent B Solvent C Solvent programming
5 L 35 ◦ C 0.25 mL/min Water Methanol 10% HCOOH in watera 89% A + 10% B 59% A + 40% B in 4.0 min 29% A + 70% B in 4.0 min 19% A + 80% B in 4.0 min 4% A + 95% B in 0.2 min 4% A + 95% B for 3.8 min 89% A + 10% B in 0.2 min 89% A + 10% B for 4.0 min
2. Experimental 2.1. Chemicals and reagents Commercial hormones and precursors standards such as, aldosterone (ALD), corticosterone (CRS), cortisol (CRL), cortisold4 (CRL-d4), deoxycorticosterone (DCRS), 11-deoxycortisol (11DCRL) and 11-deoxycortisol-d5 (11DCRL-d5), were supplied by Sigma–Aldrich (Madrid, Spain). Octadecyl-carbon (C18) SPE C18, Sílica and Strata-X SPE cartridges were obtained from Phenomenex (Madrid, Spain) and Oasis HLB from Waters (Madrid, Spain). The solvents used (all purchased in HPLC-gradient grade), included methanol, acetone, diethyl ether, ethyl, acetate, n-hexane and water, as well as the reagent formic acid were from Sigma–Aldrich (Madrid, Spain). Stock standard solutions of individual compounds were prepared at 0.010 mg/mL (CRL-d4, 11DCRL-d5), 0.10 mg/mL (CRL, 11DCRL) and at 1.0 mg/mL (ALD, CRS, DCRS) in methanol and stored at −20 ◦ C. Multicompound working standard solutions were prepared in methanol by dilution of the stock solutions and stored at −20 ◦ C. 2.2. Animals and sampling Pietrain × (Landrace × Large White), Duroc × (Landrace × Large White) and L62 (crossed of several breeds) × (Landrace × Large White) female and male pigs (barrow for Duroc and intact male pigs for the remaining breeds) were used in this study, weighing approximately 88 kg and 15 weeks of age. The procedure was carried out in a commercial farm where all relevant farmed animal welfare requirements are met. Pigs were housed in groups of 10–12 animals (stocking density of 1.0 m2 /pig; 88 kg/m2 ) and male and female pigs were kept in different stables. Saliva samples were taken at 8:00–8:30, 10:00–10:30, 12:00–12:30 and at 14:00–14:30 from animals housed in four pens (two replicates per sex) from three farms, one for each breed. A total of 48 saliva samples were collected. Saliva was collected by suspending twisted 100% cotton ropes (not chemically treated) into each pen at approximately 40 cm above from the floor for 30 min [16]. Pigs are naturally attracted to the rope and deposit oral fluids as they chew it. A period of time of 30 min was enough for 75% of the animals contacting with the rope in a stable of 25–30 animals [17]. After the exposure period, saliva samples were extracted from the rope by wringing the wet end into a plastic bag and clipping a bottom corner of the bag to drain the fluid into a tube. Then, samples were centrifuged (590 g/10 min) and were stored at −80 ◦ C until assayed.
Detection conditions Compound
Transition reactions (m/z)
CRL CRL-d4 11DCRL 11DCRL-d5 CRS DCRS ALD
363/121 367/121 347/109 352/112 347/121 373/109 361/343
a During the gradient LC separation the concentration of formic acid was constant at 0.1% (v/v).
contains a quaternary pump, auto sampler, degasser and column compartment. Chromatographic separations were performed with ˚ LC Column 100 × 2.1 mm) (Phenoa KinetexTM 2.6 m C18 (100 A, menex, Torrance, CA, USA). Chromatographic conditions are given in Table 1. A TSQ Quantum Discovery triple stage quadrupole mass spectrometer equipped to an electrospray interface from Thermo Fisher Scientific was used as detector. MS/MS analysis was performed using argon as the collision gas and nitrogen as the nebulizer gas. Quantification was performed using selected reaction monitoring (SRM) in positive mode of precursor > product ion transitions (see Table 1). Capillary voltage was set to 4.0 kV. Capillary temperature of 350 ◦ C, sheath gas and auxiliary gas pressure of 40 and 5.0 psi, Tube lens of 120 V and collision energy of 25 eV (1.5 mTorr collision cell pressure) were selected. 2.4. Extraction and purification procedure The extraction and purification procedure of the target stress hormones in pig saliva was performed by SPE with C18 and silica cartridges in series. Saliva samples (1.0 mL) were firstly passed through a C18 cartridge, which was previously activated with 3.0 mL of methanol and 1.5 mL of water. The cartridge was then washed with 0.25 mL of water, followed by 0.50 mL of water:acetone (4:1) and 0.25 mL of hexane. Then, a cartridge of silica is set in connection with the C18, which was activated beforehand with 2.0 mL of hexane followed by 1.0 mL of ethyl acetate. The hormones and precursors were then eluted from the C18-silica cartridges by passing through 6.0 mL of ethyl acetate:ethyl ether (1:1). The eluate was then evaporated to dryness, dissolved in 100 L of methanol, centrifuged at 850 g for 10 s and the supernatant was analysed by LC–MS/MS. 2.5. Analysis of variance (ANOVA) and multiple comparison test
2.3. Chromatography and mass spectrometry The liquid chromatographic system used was a Dionex Ultimate 3000 HPLC system Thermo Scientific (Madrid, Spain), which
The statistical analyses were performed with the statistical software package Statgraphics Plus v. 5.1 (Manugistics, Rockville, MD, USA). Significant differences in CRL and CRS levels amongst the
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different pig groups (separated by breed: Duroc, Pietrain and L62; and time: 0, 2.0, 4.0 and 6.0 h) were evaluated by one-way and two-way analysis of variance (ANOVA) at the 95% confidence level. A Fisher’s least significant difference (LSD) test, at a 95% confidence level, was also used to detect interactions amongst the variables. Pearson correlations among the levels of the different stress hormones in pig saliva were also performed. 3. Results and discussion One of the objectives of this work was to develop and optimize an analytical method for determining five corticosteroid hormones in pig saliva, which will serve as a tool for understanding the baseline levels in pigs at farms. Pig saliva is a very complex matrix and differs strongly with the human saliva. Human saliva is collected in special conditions (fasting conditions, 2.0 h after eating or drinking or rinse their mouths with cold water at least). These conditions could not be used for animals such as pigs in commercials farms and pig saliva samples contain feed or other interferences. Therefore a more exhaustive clean-up procedure prior to detection was required. Pre-treatment of the sample, method characterization and possible matrix effects were evaluated using spiked saliva samples (15 g/L for ALD and 5.0 for g/L for CRL, CRS, 11DCRL and DCRS). Once the analytical methodology was validated, it was applied to saliva samples for setting baseline non-stress salivary corticosteroids levels at pig farms, which is the second objective. 3.1. Optimisation of liquid chromatographic separation Chromatographic conditions were optimised for the simultaneous determination of CRL, CRS, 11DCRL, DCRS and ALD. To our knowledge, some studies about the determination of several hormones in different biological samples have been published.
Nevertheless none of them selected the target analytes [18,19]. After assaying different mobile phase systems and gradients, the mobile phase water/methanol/10% formic acid was selected (Table 1). Good chromatography is important for the analysis of steroids to ensure the adequate separation of isobaric compounds i.e. with the same mass to charge ratio (11DCRL and CRS), which all share the same nominal molecular weight and are indistinguishable in the mass spectrometer. For this reason different HPLC ˚ LC Colcolumns including a KinetexTM 2.6 m Biphenyl (100 A, ˚ LC Column 100 × 2.1 mm) umn 50 × 2.1 mm)TM , 2.6 m C18 (100 A, ˚ LC Column 100 × 4.6 mm, Ea) from and Luna® 3 m C18(2) (100 A, ˚ LC Phenomenex were tested. Only with the 2.6 m C18 (100 A, Column 100 × 2.1 mm) LC column the separation between the target isobaric compounds (11DCRL and CRS) was completely in only 20 min. The greater efficiency of the selected column increases peak resolution and improves sensitivity by giving sharp peaks with better signal to noise characteristics. The optimised conditions described above allowed the elution of hormones and their precursors within 20 min (Fig. 1). 3.2. Sample treatment The most used saliva sample treatment strategies comprise protein precipitation (PP), solid-phase extraction (SPE) and liquid–liquid extraction (LLE) [20]. Therefore, the first step of our research was to test these procedures. 3.2.1. Precipitation of salivary proteins Precipitation of proteins is a pre-treatment step for determination of glucocorticoids as CRL in biological fluids by other authors [21,22]. In this study, different protein precipitants were tested to obtain a cleaner sample and to reduce interfering substances. In this way acetonitrile acidified with 0.50% (v/v) acetic and 10% (v/v)
Fig. 1. LC–MS/MS chromatograms: A) standard chromatogram at 50 g/L (except DCRS at 25, and ALD at 100 g/L), B) saliva extract, and C) SRM chromatogram for 1: ALD, 2: CRL, 3: CRS, 4: 11DCRL, 5: DCR.
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trichloroacetic acid in cold acetone were evaluated. The method was carried out according to Jönsson et al. [22], both precipitants were added at 1.0 mL of spiked saliva sample and after 10 min at room temperature were centrifuged at 690 × g for 15 min. Then, the supernatant was evaporated in a nitrogen flow and dissolved in 100 L methanol for LC–MS/MS analysis. In this way, the final extract contained numerous smaller particles from the complex saliva sample, which could not be separated by centrifugation or filtering (on nylon and PTFE filter). The extract was not able to LC–MS/MS analysis due to it higher suspension viscosity of the final extract and therefore, only the step of protein precipitation was not enough or suitable for cleaning the extract from pig samples. 3.2.2. Extraction Because of this drawback obtained with the protein precipitation, two extraction procedures were assayed: 3.2.2.1. Liquid–liquid extraction. Recent studies showed that LLE is an effective sample preparation technique for analysis of steroids. Jensen et al. [23] and Matsui et al. [24] developed a method based on LLE followed by evaporation to dryness and reconstitution in an organic solvent for the determination of salivary CRL. According to these authors, LLE was carried out by adding 2.0 mL ethyl acetate to 1.0 mL of a spiked saliva sample. The resulting mixture was subsequently shaken for 45 min on a horizontal shaker. The samples were centrifuged for 5 min at 3500 × g and then the supernatant was evaporated to dryness under nitrogen. Finally, the residue was redissolved in 100 L methanol but a very complex extract was again obtained. 3.2.2.2. SPE extraction. SPE was frequently developed for the extraction of glucocorticoids from biological fluids. De Palo et al. [25], managed to solve the quantitative extraction of human saliva samples for measurement of CRL and cortisone using a C18. AbuRuz et al. [26] and Oledzka et al. [27] used HLB cartridges for the extraction of prednisolone, CRS and CRL from plasma and urine samples, observing quantitative recoveries. Han et al. [28] described an extraction procedure for 6-hydroxycortisol and free CRL based on Strata-X cartridges and provided recoveries of more than 93% for both analytes. In the present study, Strata-C18, Strata-X and HLB cartridges were tested. Based on the literature, several organic solvents were evaluated for conditioning, clean up and elution of cartridges. Based on the previous studies, the cartridges were first conditioned by washing with methanol and water, and the saliva sample was then loaded onto the cartridge. After a further rinse with water, followed by water:acetone (4:1) and hexane, the analytes were eluted by passing through ethyl acetate:ethyl ether (1:1). Finally, the eluate was evaporated to dryness, dissolved in 100 L of methanol. Volumes of the solvents were optimized in order to obtain the highest recoveries. Although SPE extracts were more suitable to detect by LC–MS/MS than the obtained with PPT and LLE, they still showed impurities. For this reason in the present work an additional cleanup step was added. 3.2.3. Clean up Strata-Silica cartridge was connected in series with each cartridge evaluated above (C18, Strata-X and HLB). Therefore only activation step for silica cartridge was optimized. In this way, after wash step, according to the procedure described above, the silica cartridge was inserted (previously activated with hexane followed by ethyl acetate) and the analytes were then eluted from the two cartridges connected in series. In this way for the first time, quantitative recoveries were obtained (Table 2a). The best results were achieved for C18 and silica cartridges in series. Therefore, these
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cartridges were selected to the extraction and purification of stress hormones and precursors under study from pig saliva. 3.3. Performance of the analytical method Molecules originating from the sample matrix that coelute with the compound(s) of interest can interfere with the ionization process in the mass spectrometer, causing ionization suppression or enhancement [29,30] the so-called matrix effect. In order to compensate for the alteration in signal and method fluctuations, some internal standards with similar molecular properties as the analytes under study (CRL-d4 and 11CRL-d5) were tested. The post-extraction addition technique is usually used to determine the degree of matrix effects on an LC–ESI–MS/MS method. The difference in response between the post-extraction sample and the pure solution divided by the pure solution response determines the degree of matrix effect occurring to the analyte in question under chromatographic conditions [31]. In the present study, the matrix effect was determined as around −30% for compounds under study and CRL-d4, representing a loss of 30% of the target analytes signal (ion suppression) due to alterations in ionization efficiency. Nevertheless, 11CRL-d5 was suitable internal standard only for 11CRL and different degrees of matrix effects were found for the other target analytes. CRL-d4 was then selected as internal standard. Method performance was assessed by evaluating several quality parameters of the method such as recovery values, repeatability, reproducibility, linearity and limits of detection and quantification. For this purpose, saliva samples were previously fortified with the target analytes at different levels and treated following the experimental conditions described in Section 2.3. For the assessment of the linearity of the method, a saliva sample was fortified with the target analytes at different levels by triplicate (n = 6, ranging from 0.40 to 25 g/L) and subjected to the optimized protocol. About 10 L of 500 g/L of internal standard was added to the fortified saliva samples and internal calibration was carried out. To statistically validate the regression analysis, the linearity was verified by the Mandel fitting test (P = 99%) [32]. Acceptable linearity was obtained for each compound evaluated with a correlation coefficient >0.9920 (Table 2A). To confirm that the matrix effects are corrected, recoveries of each compound were evaluated from the ratio between the slope of the calibration curve of the spiked samples and the slope of the calibration line corresponding to the hormones standards. The concentrations considered for both calibration lines ranged from 0.40 to 25 g/L (n = 6 in triplicate). The recoveries obtained ranged from 74% to 90% with the exception of ALD (60%) (Table 2B). Repeatability (within-day precision) and reproducibility (between-day precision) studies were calculated by analysing a saliva sample. In order to determine the repeatability of the method, replicate analysis were carried out on the same day (n = 5) and the reproducibility was tested over 3 consecutive days by performing five repeated analysis each day (n = 15), respectively. As can be seen in Table 2B, the relative standard deviations (RSD%) for both, repeatability and reproducibility, was lower than 11%. These RSD values testify to the excellent ruggedness of the method. Limits of detection (LOD) and quantification (LOQ) were calculated in an unfortified saliva sample following the signal-to-noise criteria (S/N = 3 and S/N = 10 for LODs and LOQs, respectively) [33]. The estimated LODs ranged from 0.020 to 0.10 g/L, whereas LOQs ranged from 0.050 to 0.31 g/L for the tested analytes (Table 2B). 3.4. Determination of stress hormones and precursors in pig saliva Once the analytical methodology was validated, it was applied to saliva samples collected in different pig farms. Forty-eight saliva
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Table 2 Recoveries comparing different SPE cartridges (% ± SD) in (A), and performance of the optimized method with the C18 cartridge for the determination of stress hormones and their precursors in pig saliva (B). (A) Compound
C18
Strata-X
HLB
CRL 11DCRL CRS DCRS ALD
74 ± 5.3 86 ± 8.1 90 ± 6.9 59 ± 4.3 74 ± 8.3
85 ± 8.4 <50 59 ± 7.3 <50 <50
55 ± 5.8 <50 66 ± 6.2 <50 <50
(B) Compound
Recoverya (%R)
Repeatabilitya (%RSD)
Reproducibilityb (%RSD)
Linear dynamic rangec (g/L)
Determination coefficient (r2 )
LOD (g/L)
LOQ (g/L)
CRL 11DCRL CRS DCRS ALD
74 86 90 60 74
5.3 6.9 8.1 4.3 8.3
7.3 7.5 10.7 6.6 9.4
0.40–10.0 0.5–10.0 0.75–10.0 0.5–10.0 2.0–25.0
0.9987 0.9991 09994 0.9920 09954
0.020 0.020 0.030 0020 0.100
0.050 0.050 0.080 0.050 0.30
a b c
n = 5. n = 15. n = 6.
samples (three breeds (Duroc, L62 or Pietrain) × two sex (male or female) × four sampling times (0, 2.0, 4.0 and 6.0 h) in duplicate) were analysed. A chromatogram of a saliva sample is shown in Fig. 1B. Fig. 2 shows the results for the selected saliva samples. As can be seen, only CRL and CRS were detected in the sampling saliva samples of pigs at farms. No significant differences in hormone levels were found between sexes (two integrated samples from 12 pigs were compared per sex). Data were then pooled for comparison at 0, 2.0, 4.0 and 6.0 h (four integrated samples per time) and no significant differences were found for any of the pig breeds although the concentrations of CRL had a clear gradient for breeds: Duroc > L62 = Pietrain. It was also found that significantly higher basal plasma CRL levels were detected in Erhualian breed [34,35]. This breed is well-known for their high fertility and superior meat quality with high intramuscular fat content and these characteristics are common in Duroc breed. The breed differences found in basal CRL levels were suggested to reflect the fundamental
differences in adrenocortical function [36]. As it was also previously documented, the baseline salivary CRL concentrations in pigs differ with housing conditions [37,38]. In the present study, CRL concentrations were within the same range as has been found previously for pigs housed under similar conditions (sampling time and under intensive conditions in a barren and restricted environment) [37,38]. Similar non-stress levels of CRL were found also by Geverink et al. [39] and Schönreiter and Zanella [40] in their studies about responses to different stressors. No differences were also found for CRS for among the breeds, but their levels trend to be higher than those of CRL (Fig. 2). Usually, the levels of CRS were clearly higher in the central hours of the morning as compared to those of CRL. Pearson correlations among the levels of the different stress hormones in pig saliva were found to be significant for CRL and CRS (n = 48, r = 0.524, P < 0.0001), which is a clear fact that they have similar responses to stress, indicating that both hormones could be stress indicators, and therefore CRS could be also used as biomarker of non-stress in saliva. Based on the results obtained as well as the results obtained by other authors, CRL and CRS levels of, respectively, 3.0 and 4.0 g/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farms. Stressors increasing their levels could be of many different origins, e.g. ecological (acute environmental changes, nutrition or shelters absence, temperature variations, etc.), socio-biological (unstable social hierarchy), health (fever, infection, injury, surgery, etc.), transport and many others. All these stressors trigger stress response – a complex of physiological, endocrine, metabolic and behaviour reactions protecting organism prior to the injurious effect of stressors (emergency life-history) [4].
4. Conclusions
Fig. 2. CRL and CRS levels compared regarding breed (Duroc, D; L, L62; and Pietrain, P). No differences at 95% probability were found for both pig sexes (male and female), but also for time between 8:00 and 14:30 h (time 0 = 8:00–8:30, time 2 = 10:00–10:30, time 4 = 12:00–12:30 and time 6 = 14:00–14:30). For these reasons, data were pooled.
An analytical method for the determination of five corticosteroids (DCRS, CRS, ALD, 11DCRL and CRL), as porcine stress biomarkers in saliva was developed and optimized. Quantitative recoveries were obtained using SPE with C18 and silica cartridges at the same time in order to perform extraction and clean up in the same step. LC–MS/MS with ESI in positive mode was used to detect the target corticosteroids. This method was applied to saliva samples from three breeds of pigs (Duroc, L62 and Pietrain) at farms in order to establish basal levels of the target corticosteroids. Since only CRL and CRS were detected in the selected samples and a significant correlation was detected between their levels, we could
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