Research article Received: 18 August 2014,
Revised: 2 October 2014,
Accepted: 17 October 2014
Published online in Wiley Online Library: 2 December 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3395
Simultaneous quantification of leukotrienes and hydroxyeicosatetraenoic acids in cell culture medium using liquid chromatography/tandem mass spectrometry Ayako Furugena, Hiroaki Yamaguchib* and Nariyasu Manob ABSTRACT: Leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) are important bioactive lipid mediators that participate in various pathophysiological processes. To advance understanding of the mechanisms that regulate these mediators in physiological and pathological processes, an analytical method using liquid chromatography/tandem mass spectrometry for the simultaneous quantification of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE in cell culture media was developed. A Supel™-Select HLB solid-phase extraction cartridge was used for sample preparation. The compounds were separated on a C18 column using gradient elution with acetonitrile–water–formic acid (20:80:0.1, v/v/v) and acetonitrile–formic acid (100:0.1, v/v). The calibration curves of LTB4, LTD4, LTE4 and HETEs were linear in the range of 0.025–10 ng/mL, and the calibration curve of LTC4 was linear in the range of 0.25–10 ng/mL. Validation assessment showed that the method was highly reliable with good accuracy and precision. The stability of LTs and HETEs was also investigated. Using the developed method, we measured LTs and HETEs in the culture supernatant of the human mast cell line HMC-1. The present method could facilitate investigations of the mechanisms that regulate the production, release and signaling of LTs and HETEs. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: leukotriene; hydroxyeicosatetraenoic acid; lipoxygenase; liquid chromatography/tandem mass spectrometry; cell culture medium
Introduction
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Eicosanoids, including prostanoids (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs), are lipid mediators. They are derived from polyunsaturated fatty acids such as arachidonic acid (AA). Eicosanoids are generated through cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P-450 (CYP) and nonenzymatic pathways (Wang and Dubois, 2010). AA is released from the cell membrane into the cytoplasm by phospholipase A2. Although COX-derived AA metabolites, PGs, have been the main focus of study to date, LTs and HETEs are also implicated in various physiological and pathological processes, such as inflammation, asthma, cancer and cardiovascular disease (Montuschi et al., 2007; Peters-Golden and Henderson, 2007; Pidgeon et al., 2007). LTs are formed through the LOX pathway. The main LOXs in humans are 5-, 12- and 15-LOX. In the 5-LOX pathway, AA is metabolized to the unstable LTA4. LTA4 is further converted to LTB4 by LTA4 hydrolase or to LTC4 by LTC4 synthase. Studies suggest that LTB4 and LTC4 are released from cells by a specific transport system (multidrug resistance-associated proteins; Keppler, 2011; Leier et al., 1994; Rius et al., 2008). The released LTC4 is converted to LTD4 and LTE4 by sequential amino acid hydrolysis. LTC4, LTD4 and LTE4 are defined as cysteinyl leukotrienes (CysLTs). The LTs exert their biological effects by binding to cell surface receptors. 5-HETE is formed by 5-LOX and free radical oxidation of AA. 12-HETE is a product of 12-LOX, CYP and radical oxidation of AA. 15-HETE is formed by 15-LOX and CYP (Murphy et al., 2005). Although 8-LOX is present in mice,
Biomed. Chromatogr. 2015; 29: 1084–1093
human 8-LOX has not been reported. Because 8-HETE has been detected in human samples (Mal et al., 2011), 8-HETE was measured in the present study. To understand the roles and regulatory mechanisms of eicosanoids, an analytical method for simultaneous quantification is needed. The measurement of eicosanoids in biological fluids such as plasma and tissue samples is important for studying the roles of eicosanoids in physiological and pathological processes. In addition, cell culture is a widely used and useful tool for evaluating the action, synthesis, transport and inactivation of eicosanoids in detail. Various methods, including high-performance liquid chromatography, gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry (LC/MS),
* Correspondence to: H. Yamaguchi, Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan. Email: yamaguchi@hosp. tohoku.ac.jp a
Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
b
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan Abbreviations used: AA, arachidonic acid; COX, cyclooxygenase; CYP, cytochrome P-450; CysLT, cysteinyl leukotriene; HETE, hydroxyeicosatetraenoic acid; LLOQ, lower limit of quantification; LT, leukotriene; LOX, lipoxygenase; PG, prostanoids; SRM, selected reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
Simultaneous quantification of LTs and HETEs have already been developed to measure several eicosanoids (Tsukamoto et al., 2002; Yue et al., 2004, 2007). Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been widely used for the quantification of various eicosanoids because it has high sensitivity and specificity and enables simultaneous analysis. Numerous analytical methods to detect LTs and HETEs in biological samples, including tissues, plasma and other biological fluids, have been developed (Cao et al., 2008; Golovko and Murphy, 2008; Miller et al., 2009; Sterz et al., 2012; Strassburg et al., 2012; Willey et al., 2008; Yang et al., 2006, 2009; Zhang et al., 2007). However, fewer methods are available for quantifying LTs and HETEs at the cellular level. Eicosanoids are present at low levels, and they exert their biological effects at nanomolar concentrations (Tager and Luster, 2003; Heise et al., 2000). Thus, sensitive and accurate analytical methods are required. Martin-Venegas et al. have developed a simultaneous quantification method for 15 lipid mediators, including PGs, LTs, HETEs, epoxyeicosatrienoic acids, dihydroxyeicosatetraenoic acids and 13-hydroxyoctadecadienoic acid, in murine 3T6 cell supernatants (Martin-Venegas et al., 2011). Although LTB4 and LTE4 in the LT family were included in the method, LTC4 and LTD4 were not measured. Furthermore, the method required a large volume of sample (5 mL), and the recovery rates ranged from 22.5 to 99.9%. Le Faouder et al. have described a rapid quantification method for 26 mediators, including pro-inflammatory and pro-resolving mediators, in Caco2 cell supernatants and colonic tissue (Le Faouder et al., 2013). CysLTs were not included, although LTB4 was measured. In the present study, we measured mainly LOX-related AA metabolites. A method for quantifying the major AA-derived LTs and HETEs could be useful for studies in various cell lines. However, to our knowledge, there are not many methods for the simultaneous quantification of LTs and HETEs in cultured cells. Eicosanoids are important mediators, and a robust quantitative method for the analysis of cellular samples could be useful for examining the roles and regulatory mechanisms of eicosanoids. In this study, we developed a simple and robust LS/MS/MS-based analytical method for the simultaneous quantification of eight AA metabolites, LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HET, and
15-HETE, in cell culture media. We applied the method to the human mast cell line HMC-1.
Experimental Chemicals Leukotriene B4 (LTB4, purity ≥97%), leukotriene C4 (LTC4, purity ≥97%), leukotriene D4 (LTD4, purity ≥97%), leukotriene E4 (LTE4, purity ≥97%), 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid [5(S)-HETE, purity ≥98%], 8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid [8(S)-HETE, purity ≥98%], 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE, purity ≥98%], 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15(S)-HETE, purity ≥98%], LTB4-d4 (purity ≥97%), LTC4-d5 (purity ≥97%), LTD4-d5 (purity ≥97%), LTE4-d5 (purity ≥97%), 5(S)-HETE-d8 (purity ≥98%), 12(S)-HETE-d8 (purity ≥98%), 15(S)-HETE-d8 (purity ≥98%) and AA were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The calcium ionophore A23187 was purchased from Sigma Aldrich (St Louis, MO, USA). All organic solvents and other chemicals were purchased from Wako (Tokyo, Japan).
LC/MS/MS Chromatographic separation was carried out using a Shimadzu Prominence 20A System (Shimadzu, Kyoto, Japan) with a Shiseido Capcell Pak C18 MGII column (2.0 ×150 mm, 5 μm). The mobile phase flow rate was 0.2 mL/min. Mobile phase A consisted of acetonitrile–water–formic acid (20:80:0.1, v/v/v), and mobile phase B consisted of acetonitrile–formic acid (100:0.1, v/v). Mobile phase B was increased from 0 to 100% in a linear gradient over 6 min and maintained at 100% until 10 min. Mobile phase B was then decreased to 0% from 10 to 11 min and maintained at 0% until 16 min. The column temperature was maintained at 40°C. The injection volume was 25 μL. The overall run time was 16 min. Negative ion electrospray tandem mass spectrometric analysis was carried out using an Applied Biosystems (Foster City, CA, USA) API 3200™ LC/MS/MS System at unit resolution with selected reaction monitoring (SRM). SRM was performed by monitoring the transitions summarized in Table 1. The parameter settings were as follows: source temperature of 600°C, spray voltage of 4000 V, curtain gas of 25 psi, ion source gas 1 of 55 psi, ion source gas 2 of 70 psi, collision gas of 6 arbitrary units and dwell time of 65 ms per ion. Data were acquired and analyzed using Analyst software (version 1.5) (Applied Biosystems).
Table 1. Selected reaction monitoring (SRM) parameters for the determination of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) Analyte LTB4 LTB4-d4 (IS for LTB4) LTC4 LTC4-d5 (IS for LTC4) LTD4 LTD4-d5 (IS for LTD4) LTE4 LTE4-d5 (IS for LTE4) 5-HETE 5-HETE-d8 (IS for 5-HETE) 8-HETE 12-HETE 12-HETE-d8 (IS for 8-HETE and 12-HETE) 15-HETE 15-HETE-d8 (IS for 15-HETE)
Transition (m/z) 335 339 624 629 495 500 438 443 319 327 319 319 327 319 327
→195 →197 →272 →272 →177 →177 →333 →338 →115 →116 →155 →179 →184 →219 →226
DP (V)
EP (V)
55 55 50 50 45 45 35 35 35 35 30 35 35 25 25
4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.0 3.5 3.5 4.5 4.5
CE (V) 28 28 28 28 28 28 20 20 18 18 18 26 26 14 14
CEP (V) 16 16 44 44 22 22 40 40 24 24 18 16 16 32 32
CXP (V) 1 1 3 3 1 1 3 3 1 1 3 3 3 1 1
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DP, Declustering potential; EP, Entrance potential; CE, Collision energy; CEP, Collision cell entrance potential; CXP, Collision cell exit potential.
A. Furugen et al. Standard stock solutions
Solid-phase extraction
Standard stock solutions containing mixtures of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE were prepared in methanol (2.5, 5, 25, 50, 250 and 500 ng/mL). An internal standard (IS) stock solution containing LTB4-d4 (20 ng/mL), LTC4-d5 (100 ng/mL), LTD4-d5 (20 ng/mL), LTE4-d5 (40 ng/mL), 5-HETE-d8 (20 ng/mL), 12-HETE-d8 (20 ng/mL) and 15-HETE-d8 (20 ng/mL) was prepared in methanol. All stock solutions were stored at 80°C.
A Supel™-Select HLB solid-phase extraction (SPE) cartridge (30 mg/mL) (Sigma Aldrich) was used for the extraction of LTs and HETEs. To each sample of culture medium (1 mL), 100 μL of a mixture of ISs was added. The sample was then added to the SPE cartridge, which was preconditioned with 1 mL of methanol and 1 mL of water. After the sample was loaded, the cartridge was washed with 1 mL of methanol–water (15:85, v/v). The LTs and HETEs were eluted with 1 mL of methanol. The eluate was dried under a nitrogen gas stream, and the residue was reconstituted in 50 μL of acetonitrile–water (1:1, v/v).
Cell culture The human mast cell line HMC-1 was kindly provided by Joseph H. Butterfield (Mayo Clinic, Rochester, MN, USA). HMC-1 cells were kept in Iscove’s modified Dulbecco’s medium (Gibco/Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (Wako), 1% penicillin–streptomycin (Sigma Aldrich) and 1.2 mM alpha-thioglycerol (Sigma Aldrich) at 37°C under 5% CO2. For induction of eicosanoid production, HMC-1 cells 7 (1 × 10 cells) were treated with A23187 (10 μM) and AA (10 μM). After treatment, the cell suspension was centrifuged at 1300g for 2 min at 4°C. The medium was collected and stored at 80°C.
Method validation Linearity and LLOQ. Calibration solutions were prepared from stock solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 2.5, 5, and 10 ng/mL in 1 mL of blank medium. The samples were extracted as described in Solid-phase extraction and analyzed. Calibration curves were constructed by plotting the peak area ratio (standard to IS) versus the nominal concentration and were fitted using least-squares regression with 1/x weighting. The lower limit of quantification (LLOQ) was defined
Figure 1. Structures of leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs) (A), and their internal standards (B).
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Figure 2. Product ion mass spectra of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE.
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Simultaneous quantification of LTs and HETEs as the concentration with a signal-to-noise ratio of at least 10 and acceptable precision and accuracy data [relative standard deviation (RSD) and relative error (RE) 80%). On the other hand, the recovery rate for LTC4 ranged from 52.5 to 59%. The extraction efficiency for LTC4 was low when compared with that of the other compounds, although the reason for this was not clear. Chappell et al. (2011) reported good recovery efficiency of LTs from sputum using an Oasis MAX cartridge (mixed-mode anion-exchange and reversed-phase). These results suggest that extraction efficiency varies and depends on many factors, including the type of SPE cartridge and the chemical properties of the eicosanoids
Calibration curve The present method covered a linearity range of 0.25–10 ng/mL for LTC4 and ranges of 0.025–10 ng/mL for LTB4, LTD4, LTE4, 5HETE, 8-HETE, 12-HETE and 15-HETE. The correlation coefficients (r2) were >0.99. Typical standard curves are summarized in Table 2. Although the analytical run time was comparable, the minimum concentrations of calibration curves of LTB4, LTD4, LTE4 and HETEs were superior to those obtained with previously described methods (Le Faouder et al., 2013; Martin-Venegas et al., 2011). Le Faouder et al. (2013) reported the LLOQs of LTB4, 5-HETE, 8-HETE, 12-HETE and 15-HETE to be 0.12, 0.98, 0.98, 7.81 and 0.98 ng/mL, respectively. Martin-Venegas et al. (2011) reported the liner ranges of the calibration curves to be 0.1–200 ng/mL for LTB4, 5-HETE, 12-HETE and 15-HETE, and 1–200 ng/mL for LTD4. Improvement of recovery rates (as described in the ‘Recovery’ section) may affect the improvement of sensitivity. The sensitivity of LTC4 was low compared with the other compounds. Although the reason for low sensitivity of LTC4 is not clear, the low recovery Table 5. Short-term stability of LTs and HETEs in the medium
Short-term stability (percentage remaining) Room temperature 2.5 ng/mL (n =3) LTB4 LTC4 LTD4 LTE4 5-HETE 8-HETE 12-HETE 15-HETE
4 24 4 24 4 24 4 24 4 24 4 24 4 24 4 24
h h h h h h h h h h h h h h h h
88.3 55.3 66.0 10.6 63.1 13.6 66.0 20.3 83.2 61.5 98.1 79.9 92.3 71.6 92.7 81.9
±2.3 ±1.3 ±10.6 ±1.9 ±2.2 ±2.0 ±2.0 ±2.0 ±6.5 ±2.0 ±1.4 ±1.9 ±5.4 ±2.1 ±1.4 ±2.7
10 ng/mL (n =3) 87.7 ±1.6 63.7 ±1.1 63.2 ±2.1 18.7 ±3.9 65.8 ±1.0 20.7 ±0.6 72.1 ±2.8 25.5 ±0.8 85.7 ±4.7 74.6 ±1.9 85.8 ±8.0 77.2 ±3.7 86.0± 4.0 75.8 ±6.1 89.8 ±0.7 87.9 ±2.8
4°C 2.5 ng/mL (n =3) 98.0 97.2 96.4 90.8 97.2 90.3 97.3 92.7 83.2 69.5 98.8 90.3 94.1 81.7 96.5 87.3
±3.2 ±1.7 ±9.5 ±6.2 ±1.4 ±2.6 ±1.3 ±2.6 ±1.1 ±3.0 ±3.1 ±6.4 ±2.2 ±3.8 ±5.4 ±0.8
10 ng/mL (n =3) 97.7 99.1 102.0 94.1 100.6 95.7 102.7 98.7 90.1 89.4 88.7 79.3 91.9 84.2 92.6 91.2
±2.1 ±3.4 ±5.2 ±4.0 ±2.7 ±0.6 ±2.1 ±2.2 ±1.3 ±7.4 ±3.5 ±2.2 ±2.2 ±2.4 ±3.0 ±1.1
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Each value represents the mean ± SD.
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A. Furugen et al. (Martin-Venegas et al., 2014). In this study, an HLB cartridge was selected because it yielded relatively good recovery rates for all compounds and relatively good cleanup results.
Precision and accuracy The intraday precision, interday precision and accuracy were investigated. The results are summarized in Table 4. The intraday
precision ranged from 2.0 to 9.4%, and the accuracies were 4.9–14.2%, except for LLOQ. The intraday precision of LLOQ ranged from 3.2 to 19.8%, and the accuracies of LLOQ were 19.9 to 19.7%. The interday precision ranged from 1.2 to 11.6%, and the accuracies were 9.5 to 9.3%, except for LLOQ. The interday precision of LLOQ ranged from 7.9 to 14.1%, and the accuracies of LLOQ were 10.5 to 11.3%. The accuracy and precision data were acceptable. Stability
Table 6. Long-term stability of LTs and HETEs Long-term stability (percentage remaining) 2.5 ng/mL (n =3) 2 weeks 4 weeks LTC4 2 weeks 4 weeks LTD4 2 weeks 4 weeks LTE4 2 weeks 4 weeks 5-HETE 2 weeks 4 weeks 8-HETE 2 weeks 4 weeks 12-HETE 2 weeks 4 weeks 15-HETE 2 weeks 4 weeks LTB4
98.7 92.7 87.9 90.7 90.4 82.7 93.3 87.7 85.6 80.9 85.7 85.6 85.6 81.2 84.0 85.1
±2.7 ±2.5 ±4.0 ±4.4 ±2.8 ±3.7 ±6.3 ±4.0 ±3.0 ±4.8 ±9.1 ±5.0 ±5.9 ±1.4 ±4.2 ±5.7
Each value represents the mean ± SD.
10 ng/mL (n =3) 99.0 96.5 90.8 96.1 94.5 88.5 97.3 93.6 97.5 80.3 89.5 87.9 94.8 86.3 89.9 87.9
±2.6 ±1.9 ±8.8 ±10.3 ±1.7 ±4.4 ±5.1 ±1.9 ±7.5 ±7.0 ±5.7 ±5.5 ±12.2 ±3.7 ±5.7 ±6.2
Because there is little information regarding the stability of LTs and HETEs in cell culture media, short-term and long-term stabilities were examined. To evaluate the short-term stability of LTs and HETEs, the amounts remaining after the addition of analytes to the medium were measured. LTs, particularly CysLTs, were unstable at room temperature. However, the remaining level of all analytes was >80% after 4 h at 4°C (Table 5). Because sample preparation takes
Revised: 2 October 2014,
Accepted: 17 October 2014
Published online in Wiley Online Library: 2 December 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3395
Simultaneous quantification of leukotrienes and hydroxyeicosatetraenoic acids in cell culture medium using liquid chromatography/tandem mass spectrometry Ayako Furugena, Hiroaki Yamaguchib* and Nariyasu Manob ABSTRACT: Leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) are important bioactive lipid mediators that participate in various pathophysiological processes. To advance understanding of the mechanisms that regulate these mediators in physiological and pathological processes, an analytical method using liquid chromatography/tandem mass spectrometry for the simultaneous quantification of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE in cell culture media was developed. A Supel™-Select HLB solid-phase extraction cartridge was used for sample preparation. The compounds were separated on a C18 column using gradient elution with acetonitrile–water–formic acid (20:80:0.1, v/v/v) and acetonitrile–formic acid (100:0.1, v/v). The calibration curves of LTB4, LTD4, LTE4 and HETEs were linear in the range of 0.025–10 ng/mL, and the calibration curve of LTC4 was linear in the range of 0.25–10 ng/mL. Validation assessment showed that the method was highly reliable with good accuracy and precision. The stability of LTs and HETEs was also investigated. Using the developed method, we measured LTs and HETEs in the culture supernatant of the human mast cell line HMC-1. The present method could facilitate investigations of the mechanisms that regulate the production, release and signaling of LTs and HETEs. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: leukotriene; hydroxyeicosatetraenoic acid; lipoxygenase; liquid chromatography/tandem mass spectrometry; cell culture medium
Introduction
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Eicosanoids, including prostanoids (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs), are lipid mediators. They are derived from polyunsaturated fatty acids such as arachidonic acid (AA). Eicosanoids are generated through cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P-450 (CYP) and nonenzymatic pathways (Wang and Dubois, 2010). AA is released from the cell membrane into the cytoplasm by phospholipase A2. Although COX-derived AA metabolites, PGs, have been the main focus of study to date, LTs and HETEs are also implicated in various physiological and pathological processes, such as inflammation, asthma, cancer and cardiovascular disease (Montuschi et al., 2007; Peters-Golden and Henderson, 2007; Pidgeon et al., 2007). LTs are formed through the LOX pathway. The main LOXs in humans are 5-, 12- and 15-LOX. In the 5-LOX pathway, AA is metabolized to the unstable LTA4. LTA4 is further converted to LTB4 by LTA4 hydrolase or to LTC4 by LTC4 synthase. Studies suggest that LTB4 and LTC4 are released from cells by a specific transport system (multidrug resistance-associated proteins; Keppler, 2011; Leier et al., 1994; Rius et al., 2008). The released LTC4 is converted to LTD4 and LTE4 by sequential amino acid hydrolysis. LTC4, LTD4 and LTE4 are defined as cysteinyl leukotrienes (CysLTs). The LTs exert their biological effects by binding to cell surface receptors. 5-HETE is formed by 5-LOX and free radical oxidation of AA. 12-HETE is a product of 12-LOX, CYP and radical oxidation of AA. 15-HETE is formed by 15-LOX and CYP (Murphy et al., 2005). Although 8-LOX is present in mice,
Biomed. Chromatogr. 2015; 29: 1084–1093
human 8-LOX has not been reported. Because 8-HETE has been detected in human samples (Mal et al., 2011), 8-HETE was measured in the present study. To understand the roles and regulatory mechanisms of eicosanoids, an analytical method for simultaneous quantification is needed. The measurement of eicosanoids in biological fluids such as plasma and tissue samples is important for studying the roles of eicosanoids in physiological and pathological processes. In addition, cell culture is a widely used and useful tool for evaluating the action, synthesis, transport and inactivation of eicosanoids in detail. Various methods, including high-performance liquid chromatography, gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry (LC/MS),
* Correspondence to: H. Yamaguchi, Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan. Email: yamaguchi@hosp. tohoku.ac.jp a
Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
b
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan Abbreviations used: AA, arachidonic acid; COX, cyclooxygenase; CYP, cytochrome P-450; CysLT, cysteinyl leukotriene; HETE, hydroxyeicosatetraenoic acid; LLOQ, lower limit of quantification; LT, leukotriene; LOX, lipoxygenase; PG, prostanoids; SRM, selected reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
Simultaneous quantification of LTs and HETEs have already been developed to measure several eicosanoids (Tsukamoto et al., 2002; Yue et al., 2004, 2007). Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been widely used for the quantification of various eicosanoids because it has high sensitivity and specificity and enables simultaneous analysis. Numerous analytical methods to detect LTs and HETEs in biological samples, including tissues, plasma and other biological fluids, have been developed (Cao et al., 2008; Golovko and Murphy, 2008; Miller et al., 2009; Sterz et al., 2012; Strassburg et al., 2012; Willey et al., 2008; Yang et al., 2006, 2009; Zhang et al., 2007). However, fewer methods are available for quantifying LTs and HETEs at the cellular level. Eicosanoids are present at low levels, and they exert their biological effects at nanomolar concentrations (Tager and Luster, 2003; Heise et al., 2000). Thus, sensitive and accurate analytical methods are required. Martin-Venegas et al. have developed a simultaneous quantification method for 15 lipid mediators, including PGs, LTs, HETEs, epoxyeicosatrienoic acids, dihydroxyeicosatetraenoic acids and 13-hydroxyoctadecadienoic acid, in murine 3T6 cell supernatants (Martin-Venegas et al., 2011). Although LTB4 and LTE4 in the LT family were included in the method, LTC4 and LTD4 were not measured. Furthermore, the method required a large volume of sample (5 mL), and the recovery rates ranged from 22.5 to 99.9%. Le Faouder et al. have described a rapid quantification method for 26 mediators, including pro-inflammatory and pro-resolving mediators, in Caco2 cell supernatants and colonic tissue (Le Faouder et al., 2013). CysLTs were not included, although LTB4 was measured. In the present study, we measured mainly LOX-related AA metabolites. A method for quantifying the major AA-derived LTs and HETEs could be useful for studies in various cell lines. However, to our knowledge, there are not many methods for the simultaneous quantification of LTs and HETEs in cultured cells. Eicosanoids are important mediators, and a robust quantitative method for the analysis of cellular samples could be useful for examining the roles and regulatory mechanisms of eicosanoids. In this study, we developed a simple and robust LS/MS/MS-based analytical method for the simultaneous quantification of eight AA metabolites, LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HET, and
15-HETE, in cell culture media. We applied the method to the human mast cell line HMC-1.
Experimental Chemicals Leukotriene B4 (LTB4, purity ≥97%), leukotriene C4 (LTC4, purity ≥97%), leukotriene D4 (LTD4, purity ≥97%), leukotriene E4 (LTE4, purity ≥97%), 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid [5(S)-HETE, purity ≥98%], 8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid [8(S)-HETE, purity ≥98%], 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE, purity ≥98%], 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15(S)-HETE, purity ≥98%], LTB4-d4 (purity ≥97%), LTC4-d5 (purity ≥97%), LTD4-d5 (purity ≥97%), LTE4-d5 (purity ≥97%), 5(S)-HETE-d8 (purity ≥98%), 12(S)-HETE-d8 (purity ≥98%), 15(S)-HETE-d8 (purity ≥98%) and AA were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The calcium ionophore A23187 was purchased from Sigma Aldrich (St Louis, MO, USA). All organic solvents and other chemicals were purchased from Wako (Tokyo, Japan).
LC/MS/MS Chromatographic separation was carried out using a Shimadzu Prominence 20A System (Shimadzu, Kyoto, Japan) with a Shiseido Capcell Pak C18 MGII column (2.0 ×150 mm, 5 μm). The mobile phase flow rate was 0.2 mL/min. Mobile phase A consisted of acetonitrile–water–formic acid (20:80:0.1, v/v/v), and mobile phase B consisted of acetonitrile–formic acid (100:0.1, v/v). Mobile phase B was increased from 0 to 100% in a linear gradient over 6 min and maintained at 100% until 10 min. Mobile phase B was then decreased to 0% from 10 to 11 min and maintained at 0% until 16 min. The column temperature was maintained at 40°C. The injection volume was 25 μL. The overall run time was 16 min. Negative ion electrospray tandem mass spectrometric analysis was carried out using an Applied Biosystems (Foster City, CA, USA) API 3200™ LC/MS/MS System at unit resolution with selected reaction monitoring (SRM). SRM was performed by monitoring the transitions summarized in Table 1. The parameter settings were as follows: source temperature of 600°C, spray voltage of 4000 V, curtain gas of 25 psi, ion source gas 1 of 55 psi, ion source gas 2 of 70 psi, collision gas of 6 arbitrary units and dwell time of 65 ms per ion. Data were acquired and analyzed using Analyst software (version 1.5) (Applied Biosystems).
Table 1. Selected reaction monitoring (SRM) parameters for the determination of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) Analyte LTB4 LTB4-d4 (IS for LTB4) LTC4 LTC4-d5 (IS for LTC4) LTD4 LTD4-d5 (IS for LTD4) LTE4 LTE4-d5 (IS for LTE4) 5-HETE 5-HETE-d8 (IS for 5-HETE) 8-HETE 12-HETE 12-HETE-d8 (IS for 8-HETE and 12-HETE) 15-HETE 15-HETE-d8 (IS for 15-HETE)
Transition (m/z) 335 339 624 629 495 500 438 443 319 327 319 319 327 319 327
→195 →197 →272 →272 →177 →177 →333 →338 →115 →116 →155 →179 →184 →219 →226
DP (V)
EP (V)
55 55 50 50 45 45 35 35 35 35 30 35 35 25 25
4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.0 3.5 3.5 4.5 4.5
CE (V) 28 28 28 28 28 28 20 20 18 18 18 26 26 14 14
CEP (V) 16 16 44 44 22 22 40 40 24 24 18 16 16 32 32
CXP (V) 1 1 3 3 1 1 3 3 1 1 3 3 3 1 1
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DP, Declustering potential; EP, Entrance potential; CE, Collision energy; CEP, Collision cell entrance potential; CXP, Collision cell exit potential.
A. Furugen et al. Standard stock solutions
Solid-phase extraction
Standard stock solutions containing mixtures of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE were prepared in methanol (2.5, 5, 25, 50, 250 and 500 ng/mL). An internal standard (IS) stock solution containing LTB4-d4 (20 ng/mL), LTC4-d5 (100 ng/mL), LTD4-d5 (20 ng/mL), LTE4-d5 (40 ng/mL), 5-HETE-d8 (20 ng/mL), 12-HETE-d8 (20 ng/mL) and 15-HETE-d8 (20 ng/mL) was prepared in methanol. All stock solutions were stored at 80°C.
A Supel™-Select HLB solid-phase extraction (SPE) cartridge (30 mg/mL) (Sigma Aldrich) was used for the extraction of LTs and HETEs. To each sample of culture medium (1 mL), 100 μL of a mixture of ISs was added. The sample was then added to the SPE cartridge, which was preconditioned with 1 mL of methanol and 1 mL of water. After the sample was loaded, the cartridge was washed with 1 mL of methanol–water (15:85, v/v). The LTs and HETEs were eluted with 1 mL of methanol. The eluate was dried under a nitrogen gas stream, and the residue was reconstituted in 50 μL of acetonitrile–water (1:1, v/v).
Cell culture The human mast cell line HMC-1 was kindly provided by Joseph H. Butterfield (Mayo Clinic, Rochester, MN, USA). HMC-1 cells were kept in Iscove’s modified Dulbecco’s medium (Gibco/Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (Wako), 1% penicillin–streptomycin (Sigma Aldrich) and 1.2 mM alpha-thioglycerol (Sigma Aldrich) at 37°C under 5% CO2. For induction of eicosanoid production, HMC-1 cells 7 (1 × 10 cells) were treated with A23187 (10 μM) and AA (10 μM). After treatment, the cell suspension was centrifuged at 1300g for 2 min at 4°C. The medium was collected and stored at 80°C.
Method validation Linearity and LLOQ. Calibration solutions were prepared from stock solutions at concentrations of 0.025, 0.05, 0.25, 0.5, 2.5, 5, and 10 ng/mL in 1 mL of blank medium. The samples were extracted as described in Solid-phase extraction and analyzed. Calibration curves were constructed by plotting the peak area ratio (standard to IS) versus the nominal concentration and were fitted using least-squares regression with 1/x weighting. The lower limit of quantification (LLOQ) was defined
Figure 1. Structures of leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs) (A), and their internal standards (B).
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Figure 2. Product ion mass spectra of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE.
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Simultaneous quantification of LTs and HETEs as the concentration with a signal-to-noise ratio of at least 10 and acceptable precision and accuracy data [relative standard deviation (RSD) and relative error (RE) 80%). On the other hand, the recovery rate for LTC4 ranged from 52.5 to 59%. The extraction efficiency for LTC4 was low when compared with that of the other compounds, although the reason for this was not clear. Chappell et al. (2011) reported good recovery efficiency of LTs from sputum using an Oasis MAX cartridge (mixed-mode anion-exchange and reversed-phase). These results suggest that extraction efficiency varies and depends on many factors, including the type of SPE cartridge and the chemical properties of the eicosanoids
Calibration curve The present method covered a linearity range of 0.25–10 ng/mL for LTC4 and ranges of 0.025–10 ng/mL for LTB4, LTD4, LTE4, 5HETE, 8-HETE, 12-HETE and 15-HETE. The correlation coefficients (r2) were >0.99. Typical standard curves are summarized in Table 2. Although the analytical run time was comparable, the minimum concentrations of calibration curves of LTB4, LTD4, LTE4 and HETEs were superior to those obtained with previously described methods (Le Faouder et al., 2013; Martin-Venegas et al., 2011). Le Faouder et al. (2013) reported the LLOQs of LTB4, 5-HETE, 8-HETE, 12-HETE and 15-HETE to be 0.12, 0.98, 0.98, 7.81 and 0.98 ng/mL, respectively. Martin-Venegas et al. (2011) reported the liner ranges of the calibration curves to be 0.1–200 ng/mL for LTB4, 5-HETE, 12-HETE and 15-HETE, and 1–200 ng/mL for LTD4. Improvement of recovery rates (as described in the ‘Recovery’ section) may affect the improvement of sensitivity. The sensitivity of LTC4 was low compared with the other compounds. Although the reason for low sensitivity of LTC4 is not clear, the low recovery Table 5. Short-term stability of LTs and HETEs in the medium
Short-term stability (percentage remaining) Room temperature 2.5 ng/mL (n =3) LTB4 LTC4 LTD4 LTE4 5-HETE 8-HETE 12-HETE 15-HETE
4 24 4 24 4 24 4 24 4 24 4 24 4 24 4 24
h h h h h h h h h h h h h h h h
88.3 55.3 66.0 10.6 63.1 13.6 66.0 20.3 83.2 61.5 98.1 79.9 92.3 71.6 92.7 81.9
±2.3 ±1.3 ±10.6 ±1.9 ±2.2 ±2.0 ±2.0 ±2.0 ±6.5 ±2.0 ±1.4 ±1.9 ±5.4 ±2.1 ±1.4 ±2.7
10 ng/mL (n =3) 87.7 ±1.6 63.7 ±1.1 63.2 ±2.1 18.7 ±3.9 65.8 ±1.0 20.7 ±0.6 72.1 ±2.8 25.5 ±0.8 85.7 ±4.7 74.6 ±1.9 85.8 ±8.0 77.2 ±3.7 86.0± 4.0 75.8 ±6.1 89.8 ±0.7 87.9 ±2.8
4°C 2.5 ng/mL (n =3) 98.0 97.2 96.4 90.8 97.2 90.3 97.3 92.7 83.2 69.5 98.8 90.3 94.1 81.7 96.5 87.3
±3.2 ±1.7 ±9.5 ±6.2 ±1.4 ±2.6 ±1.3 ±2.6 ±1.1 ±3.0 ±3.1 ±6.4 ±2.2 ±3.8 ±5.4 ±0.8
10 ng/mL (n =3) 97.7 99.1 102.0 94.1 100.6 95.7 102.7 98.7 90.1 89.4 88.7 79.3 91.9 84.2 92.6 91.2
±2.1 ±3.4 ±5.2 ±4.0 ±2.7 ±0.6 ±2.1 ±2.2 ±1.3 ±7.4 ±3.5 ±2.2 ±2.2 ±2.4 ±3.0 ±1.1
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Each value represents the mean ± SD.
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A. Furugen et al. (Martin-Venegas et al., 2014). In this study, an HLB cartridge was selected because it yielded relatively good recovery rates for all compounds and relatively good cleanup results.
Precision and accuracy The intraday precision, interday precision and accuracy were investigated. The results are summarized in Table 4. The intraday
precision ranged from 2.0 to 9.4%, and the accuracies were 4.9–14.2%, except for LLOQ. The intraday precision of LLOQ ranged from 3.2 to 19.8%, and the accuracies of LLOQ were 19.9 to 19.7%. The interday precision ranged from 1.2 to 11.6%, and the accuracies were 9.5 to 9.3%, except for LLOQ. The interday precision of LLOQ ranged from 7.9 to 14.1%, and the accuracies of LLOQ were 10.5 to 11.3%. The accuracy and precision data were acceptable. Stability
Table 6. Long-term stability of LTs and HETEs Long-term stability (percentage remaining) 2.5 ng/mL (n =3) 2 weeks 4 weeks LTC4 2 weeks 4 weeks LTD4 2 weeks 4 weeks LTE4 2 weeks 4 weeks 5-HETE 2 weeks 4 weeks 8-HETE 2 weeks 4 weeks 12-HETE 2 weeks 4 weeks 15-HETE 2 weeks 4 weeks LTB4
98.7 92.7 87.9 90.7 90.4 82.7 93.3 87.7 85.6 80.9 85.7 85.6 85.6 81.2 84.0 85.1
±2.7 ±2.5 ±4.0 ±4.4 ±2.8 ±3.7 ±6.3 ±4.0 ±3.0 ±4.8 ±9.1 ±5.0 ±5.9 ±1.4 ±4.2 ±5.7
Each value represents the mean ± SD.
10 ng/mL (n =3) 99.0 96.5 90.8 96.1 94.5 88.5 97.3 93.6 97.5 80.3 89.5 87.9 94.8 86.3 89.9 87.9
±2.6 ±1.9 ±8.8 ±10.3 ±1.7 ±4.4 ±5.1 ±1.9 ±7.5 ±7.0 ±5.7 ±5.5 ±12.2 ±3.7 ±5.7 ±6.2
Because there is little information regarding the stability of LTs and HETEs in cell culture media, short-term and long-term stabilities were examined. To evaluate the short-term stability of LTs and HETEs, the amounts remaining after the addition of analytes to the medium were measured. LTs, particularly CysLTs, were unstable at room temperature. However, the remaining level of all analytes was >80% after 4 h at 4°C (Table 5). Because sample preparation takes
