Research article Received: 31 December 2014,
Revised: 26 March 2015,
Accepted: 5 April 2015
Published online in Wiley Online Library: 5 May 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3487
A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development, validation and application in the CUMS rat model Xue-jia Zhai, Fen Chen, Chao-ran Zhu and Yong-ning Lu* ABSTRACT: Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC-MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25–12,800 ng mL1 with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: CUMS; urine; LC-MS/MS; depression; neurotransmitters
Introduction
Biomed. Chromatogr. 2015; 29: 1737–1743
* Correspondence to: Y.-n. Lu, Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China. Email: [email protected] Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China Abbreviations used: CUMS, chronic unpredictable mild stress; IEC, ionexchange chromatography; MRM, multiple reaction monitoring.
Copyright © 2015 John Wiley & Sons, Ltd.
1737
Free amino acids analysis is an important tool in metabolic research studies in a wide variety of medical and biopharmaceutical applications. Moreover, amino acid neurotransmitters in biological fluids are essential to the diagnosis and monitoring of depression and a wide variety of other metabolic disturbances in scientific research (Tsukano et al., 2014; Sales et al., 2014; Kamleh et al., 2015). Many amino acid analyzing methods have been developed in pharmacological study, using both pre-column and post-column derivatization techniques (Megias et al., 2015; Ziegler and Abel, 2014; Kvitvang et al., 2014). The traditional and most widely used approach for amino acid analysis is ion-exchange chromatography (IEC) with post-column ninhydrin derivatization, which has been in use for over 40 years and provides good linearity over a wide range, excellent separation and reproducibility, with simple sample preparation (Filee et al., 2014; Moulin et al., 2002). However, IEC suffers from long analysis time (Thiangthum et al., 2014). In addition, particularly in urine samples, the specificity of this analysis method is generally reduced by potential interferences from co-eluting compounds that derive from medications or dietary supplements (Lara et al., 2013; Tsai et al., 2014). Alternatively, pre-column derivatization with various reagents such as o-phthalaldehyde and separation by reversed-phase (RP)-HPLC achieves high sensitivity and fast analysis time but requires extensive sample preparation (Dai et al., 2014). All of the existing derivatization assays present various problems – reagent interference, instability of derivative, long preparation time and difficulties in derivatization to specific amino acids (Tomita et al., 2014; Cui et al., 2014). Therefore methods to
determinate amino acid neurotransmitters, without derivatization, utilizing liquid chromatography–tandem mass spectrometry (LC-MS/MS) have been reported in recent years, which have reduced sample preparation time and eliminated reagent-associated interferences (Le et al., 2014; Held et al., 2011). Some research groups have developed LC-MS/MS methods for analyzing amino acids in human plasma and urine (Waterval et al., 2009). Le et al. (2014) measured underivatized amino acids by LC-MS with column-switching technology, but the method was validated in human samples. Held et al. (2011) described the measurement of the amino acids in human urine in a single analytical run. However, the limit of quantification (LOQ) of this method is only 1 μmol/L. Other studies that comprise quantitative analysis of the amino acids just focus the analytes associated with an inborn error of metabolism in human body fluids or atmospheric amino acids (Waterval et al., 2009; Buiarelli et al., 2013). In some studies which comprise quantitative analysis of the amino acids in biological fluids, the procedure of sample preparation is complex or the analysis time is long (Kaspar et al., 2008; Lozanov et al., 2007).
X.-j. Zhai et al. Recent studies revealed that the contents of amino acids in urine significantly changed in a depression rat model ( Jia et al., 2013; Wang et al., 2012). However, to the best of our knowledge, the reports describing the analysis of the urinary amino acid neurotransmitters paid little attention to 4-aminohippurate, 5HIAA, hippurate, pimelate and tyramine, which are important neurotransmitters in the metabolic pathways associated with depression, such as norepinephrine and serotonin reuptake. In this study, an analysis method for the analysis of 11 neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) without derivation by LC-MS/MS was developed with simple preparation and short analysis time, high sensitivity and specificity. The method is simple in sample preparation with a short analysis time and high sensitivity and specificity, and is applicable for the analysis of metabolites in rat urine samples. The advantage of the presented method is the concurrent determination of 11 important neurotransmitters which are potential biomarkers in the progression of depression, and it represents an important starting point for future inter-comparison studies.
spectrometer with an electrospray ionization source (AB/MDSSciex, Ontario, Canada). The tandem mass spectrometer was operated under the multiple reaction monitoring (MRM) mode using an electrospray source in positive ion mode. The optimized condition consisted of a collision-activated dissociation gas of the medium, a curtain gas of 20 psi, a nebulizer gas of 20 psi, a TurboIonSpray gas of 10 psi, an ionspray voltage of +5500 V and a source temperature of 500 °C. The MRM transitions and the related optimized declustering potential, entrance potential, collision energy and collision cell exit potential for the different analytes are shown in Table 1. MRM data was acquired and the chromatograms were integrated by use of the Analyst 1.6.1 software. Chromatographic separation was performed on an XTerra RP18 column (100 × 3.91 mm, 3.5 μm, Waters Corporation USA). Solvents that constituted the mobile phase were A (0.1% aqueous acetic acid) and B (acetonitrile with 0.1% acetic acid). The elution conditions applied were: 0–1 min, 1% B; 1–9 min, 1–32% B; 9–11 min, 32–99% B; 12–14 min, 99% B. The flow-rate was 0.45 mL/min and the injection volume was 5 μL. The temperatures of the analytical column and autosampler were set at 40 and 4 °C, respectively.
Experimetal
Standard solutions
Materials 4-Aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine were purchased from Aladdin Inc. (Shanghai, China). Cortisol (as an internal standard, IS) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade acetonitrile and methanol were obtained from Tedia (Tedia, Fairfield, OH, USA). Formic acid (Tedia, USA) was >98% purity. Other reagents were analytical grade or better. The water used for LC-MS/MS was purified by use of a Milli-Q system (Millipore, Milford, MA, USA). Stock solutions of all standards (1.0 mg/mL) and of IS (30 ng mL1) were prepared in 2% aqueous fomic acid–methanol (90:10, v/v) and stored at 4 °C. The acetic acid in the solvent was to preserve the stability of the analytes.
Working solutions of the 11 standards (25, 50, 100, 200, 400, 800, 1600, 3200, 6400 and 12,800 ng mL1) were prepared in 200 μL urine by dilution of the stock solution of each analyte. The standards were prepared on the day of analysis. Sample preparation An aliquot of 20 μL of IS (cortisol; 30 ng mL1) was added to a 200 μL urine supernatant or calibrators in an EP tube. After vortexmixing for 30 s, the mixture was added with 200 μL acetonitrile to precipitate protein and the sample was vortex-mixed for 2 min and centrifuged at 6000 g for 10 min at 4 °C. The separated supernatant was evaporated to dryness on a water bath at 40 °C under a protective atmosphere of nitrogen. The residue was reconstituted with 100 μL 0.1% aqueous acetic acid–methanol (90:10, v/v).
Instrumentation and analytical conditions
Method validation
The LC-MS/MS system was a Shimadzu LC-30 AD pump (Shimadzu, Kyoto, Japan) and a SIL-30 AC autosampler (Shimadzu, Kyoto, Japan) coupled to an API QTRAP 5500 triple quadrupole mass
The assay was validated as meeting the published acceptance criteria for linearity, precision, matrix effect and stability proposed by the US Food and Drug Administration (2001). The concentrations
Table 1. Multiple reaction monitoring (MRM) transitions, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) of the analytes
1738
No.
Compounds
MRM transitions
DP (V)
EP (V)
CE (eV)
CXP (V)
1 2 3 4 5 6 7 8 9 10 11 12
4-Aminohippurate 5-HIAA Glutamate Glutamine Hippurate Pimelate Proline Tryptophan Tyramine Tyrosine Valine Cortisol
m/z 195.1 → m/z 120.1 m/z 192.2 → m/z 146.1 m/z 148.2 → m/z 130.1 m/z 147.2 → m/z 130.0 m/z 180.1 → m/z 105.1 m/z 161.1 → m/z 125.2 m/z 116.1 → m/z 70.2 m/z 205.2 → m/z 188.2 m/z 138.2 → m/z 121.1 m/z 182 → m/z 123 m/z 118.2 → m/z 72.1 m/z 363.2 → m/z 121.3
26 66 28 34 34 30 37 30 30 30 40 70
8 8 8 8 8 8 8 8 8 10 8 9
16 17 12 24 16 15 20 12 14 22 13 33
10 7 6 13 4 21 11 15 12 17 13 8
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1737–1743
A LC-MS method for determination of neurotransmitters in rat urine of analytes for matrix effect (ME) and recovery (RE) test were at three levels (30, 500 and 10,000 ng mL1, analysed six times at each concentration). The linearity was evaluated by the regression analysis of standards over the concentration range of the calibration curve. The precision were assessed using the three quality controls (QCs). The QCs were assayed six times within the same day for the determination of intraday precision and on six consecutive days in duplicate to determine inter-day precision, and the RSD was calculated. The effects of freezing and thawing on the concentrations of analytes were studied using the three QCs, which were subjected to three freeze–thaw cycles before analysis. The stability of urine QC samples at 20 °C was evaluated by concentration analysis at weekly intervals for 1 month. The stability of the stock standard solutions at 4 °C for 1 month was evaluated by comparing the response with that of the freshly prepared standard solutions. The stability of the processed samples at room temperature for 24 h was evaluated by comparing the results with the original results. In all cases, analytes were considered to be stable as long as degradation was
Revised: 26 March 2015,
Accepted: 5 April 2015
Published online in Wiley Online Library: 5 May 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3487
A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development, validation and application in the CUMS rat model Xue-jia Zhai, Fen Chen, Chao-ran Zhu and Yong-ning Lu* ABSTRACT: Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC-MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25–12,800 ng mL1 with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: CUMS; urine; LC-MS/MS; depression; neurotransmitters
Introduction
Biomed. Chromatogr. 2015; 29: 1737–1743
* Correspondence to: Y.-n. Lu, Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China. Email: [email protected] Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China Abbreviations used: CUMS, chronic unpredictable mild stress; IEC, ionexchange chromatography; MRM, multiple reaction monitoring.
Copyright © 2015 John Wiley & Sons, Ltd.
1737
Free amino acids analysis is an important tool in metabolic research studies in a wide variety of medical and biopharmaceutical applications. Moreover, amino acid neurotransmitters in biological fluids are essential to the diagnosis and monitoring of depression and a wide variety of other metabolic disturbances in scientific research (Tsukano et al., 2014; Sales et al., 2014; Kamleh et al., 2015). Many amino acid analyzing methods have been developed in pharmacological study, using both pre-column and post-column derivatization techniques (Megias et al., 2015; Ziegler and Abel, 2014; Kvitvang et al., 2014). The traditional and most widely used approach for amino acid analysis is ion-exchange chromatography (IEC) with post-column ninhydrin derivatization, which has been in use for over 40 years and provides good linearity over a wide range, excellent separation and reproducibility, with simple sample preparation (Filee et al., 2014; Moulin et al., 2002). However, IEC suffers from long analysis time (Thiangthum et al., 2014). In addition, particularly in urine samples, the specificity of this analysis method is generally reduced by potential interferences from co-eluting compounds that derive from medications or dietary supplements (Lara et al., 2013; Tsai et al., 2014). Alternatively, pre-column derivatization with various reagents such as o-phthalaldehyde and separation by reversed-phase (RP)-HPLC achieves high sensitivity and fast analysis time but requires extensive sample preparation (Dai et al., 2014). All of the existing derivatization assays present various problems – reagent interference, instability of derivative, long preparation time and difficulties in derivatization to specific amino acids (Tomita et al., 2014; Cui et al., 2014). Therefore methods to
determinate amino acid neurotransmitters, without derivatization, utilizing liquid chromatography–tandem mass spectrometry (LC-MS/MS) have been reported in recent years, which have reduced sample preparation time and eliminated reagent-associated interferences (Le et al., 2014; Held et al., 2011). Some research groups have developed LC-MS/MS methods for analyzing amino acids in human plasma and urine (Waterval et al., 2009). Le et al. (2014) measured underivatized amino acids by LC-MS with column-switching technology, but the method was validated in human samples. Held et al. (2011) described the measurement of the amino acids in human urine in a single analytical run. However, the limit of quantification (LOQ) of this method is only 1 μmol/L. Other studies that comprise quantitative analysis of the amino acids just focus the analytes associated with an inborn error of metabolism in human body fluids or atmospheric amino acids (Waterval et al., 2009; Buiarelli et al., 2013). In some studies which comprise quantitative analysis of the amino acids in biological fluids, the procedure of sample preparation is complex or the analysis time is long (Kaspar et al., 2008; Lozanov et al., 2007).
X.-j. Zhai et al. Recent studies revealed that the contents of amino acids in urine significantly changed in a depression rat model ( Jia et al., 2013; Wang et al., 2012). However, to the best of our knowledge, the reports describing the analysis of the urinary amino acid neurotransmitters paid little attention to 4-aminohippurate, 5HIAA, hippurate, pimelate and tyramine, which are important neurotransmitters in the metabolic pathways associated with depression, such as norepinephrine and serotonin reuptake. In this study, an analysis method for the analysis of 11 neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) without derivation by LC-MS/MS was developed with simple preparation and short analysis time, high sensitivity and specificity. The method is simple in sample preparation with a short analysis time and high sensitivity and specificity, and is applicable for the analysis of metabolites in rat urine samples. The advantage of the presented method is the concurrent determination of 11 important neurotransmitters which are potential biomarkers in the progression of depression, and it represents an important starting point for future inter-comparison studies.
spectrometer with an electrospray ionization source (AB/MDSSciex, Ontario, Canada). The tandem mass spectrometer was operated under the multiple reaction monitoring (MRM) mode using an electrospray source in positive ion mode. The optimized condition consisted of a collision-activated dissociation gas of the medium, a curtain gas of 20 psi, a nebulizer gas of 20 psi, a TurboIonSpray gas of 10 psi, an ionspray voltage of +5500 V and a source temperature of 500 °C. The MRM transitions and the related optimized declustering potential, entrance potential, collision energy and collision cell exit potential for the different analytes are shown in Table 1. MRM data was acquired and the chromatograms were integrated by use of the Analyst 1.6.1 software. Chromatographic separation was performed on an XTerra RP18 column (100 × 3.91 mm, 3.5 μm, Waters Corporation USA). Solvents that constituted the mobile phase were A (0.1% aqueous acetic acid) and B (acetonitrile with 0.1% acetic acid). The elution conditions applied were: 0–1 min, 1% B; 1–9 min, 1–32% B; 9–11 min, 32–99% B; 12–14 min, 99% B. The flow-rate was 0.45 mL/min and the injection volume was 5 μL. The temperatures of the analytical column and autosampler were set at 40 and 4 °C, respectively.
Experimetal
Standard solutions
Materials 4-Aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine were purchased from Aladdin Inc. (Shanghai, China). Cortisol (as an internal standard, IS) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade acetonitrile and methanol were obtained from Tedia (Tedia, Fairfield, OH, USA). Formic acid (Tedia, USA) was >98% purity. Other reagents were analytical grade or better. The water used for LC-MS/MS was purified by use of a Milli-Q system (Millipore, Milford, MA, USA). Stock solutions of all standards (1.0 mg/mL) and of IS (30 ng mL1) were prepared in 2% aqueous fomic acid–methanol (90:10, v/v) and stored at 4 °C. The acetic acid in the solvent was to preserve the stability of the analytes.
Working solutions of the 11 standards (25, 50, 100, 200, 400, 800, 1600, 3200, 6400 and 12,800 ng mL1) were prepared in 200 μL urine by dilution of the stock solution of each analyte. The standards were prepared on the day of analysis. Sample preparation An aliquot of 20 μL of IS (cortisol; 30 ng mL1) was added to a 200 μL urine supernatant or calibrators in an EP tube. After vortexmixing for 30 s, the mixture was added with 200 μL acetonitrile to precipitate protein and the sample was vortex-mixed for 2 min and centrifuged at 6000 g for 10 min at 4 °C. The separated supernatant was evaporated to dryness on a water bath at 40 °C under a protective atmosphere of nitrogen. The residue was reconstituted with 100 μL 0.1% aqueous acetic acid–methanol (90:10, v/v).
Instrumentation and analytical conditions
Method validation
The LC-MS/MS system was a Shimadzu LC-30 AD pump (Shimadzu, Kyoto, Japan) and a SIL-30 AC autosampler (Shimadzu, Kyoto, Japan) coupled to an API QTRAP 5500 triple quadrupole mass
The assay was validated as meeting the published acceptance criteria for linearity, precision, matrix effect and stability proposed by the US Food and Drug Administration (2001). The concentrations
Table 1. Multiple reaction monitoring (MRM) transitions, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) of the analytes
1738
No.
Compounds
MRM transitions
DP (V)
EP (V)
CE (eV)
CXP (V)
1 2 3 4 5 6 7 8 9 10 11 12
4-Aminohippurate 5-HIAA Glutamate Glutamine Hippurate Pimelate Proline Tryptophan Tyramine Tyrosine Valine Cortisol
m/z 195.1 → m/z 120.1 m/z 192.2 → m/z 146.1 m/z 148.2 → m/z 130.1 m/z 147.2 → m/z 130.0 m/z 180.1 → m/z 105.1 m/z 161.1 → m/z 125.2 m/z 116.1 → m/z 70.2 m/z 205.2 → m/z 188.2 m/z 138.2 → m/z 121.1 m/z 182 → m/z 123 m/z 118.2 → m/z 72.1 m/z 363.2 → m/z 121.3
26 66 28 34 34 30 37 30 30 30 40 70
8 8 8 8 8 8 8 8 8 10 8 9
16 17 12 24 16 15 20 12 14 22 13 33
10 7 6 13 4 21 11 15 12 17 13 8
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1737–1743
A LC-MS method for determination of neurotransmitters in rat urine of analytes for matrix effect (ME) and recovery (RE) test were at three levels (30, 500 and 10,000 ng mL1, analysed six times at each concentration). The linearity was evaluated by the regression analysis of standards over the concentration range of the calibration curve. The precision were assessed using the three quality controls (QCs). The QCs were assayed six times within the same day for the determination of intraday precision and on six consecutive days in duplicate to determine inter-day precision, and the RSD was calculated. The effects of freezing and thawing on the concentrations of analytes were studied using the three QCs, which were subjected to three freeze–thaw cycles before analysis. The stability of urine QC samples at 20 °C was evaluated by concentration analysis at weekly intervals for 1 month. The stability of the stock standard solutions at 4 °C for 1 month was evaluated by comparing the response with that of the freshly prepared standard solutions. The stability of the processed samples at room temperature for 24 h was evaluated by comparing the results with the original results. In all cases, analytes were considered to be stable as long as degradation was
