Chromatographia

, Volume 75, Issue 21, pp 1257–1269

The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin, Dopamine and Melatonin

Authors

  • Merisa Moriarty
    • Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research)Cork Institute of Technology
  • Aoife Lee
    • Department of Biological SciencesCork Institute of Technology
  • Brendan O’Connell
    • Department of Biological SciencesCork Institute of Technology
  • Mary Lehane
    • Department of Chemistry, PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research)Cork Institute of Technology
  • Helen Keeley
    • Child and Adolescent Mental Health Services, Health Service Executive, SouthNorth Cork Area and the National Suicide Research Foundation
    • Team Elucidate and PROTEOBIO (Mass Spectrometry Centre for Proteomic and Biotoxin Research) research groupsCork Institute of Technology
Original

DOI: 10.1007/s10337-012-2330-5

Cite this article as:
Moriarty, M., Lee, A., O’Connell, B. et al. Chromatographia (2012) 75: 1257. doi:10.1007/s10337-012-2330-5

Abstract

Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L−1; R2 = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L−1 and the LOQs of 15.4–70.5 nmol L−1 for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.

Keywords

LC–MS/MSHybridSPE-PrecipitationSerotoninDopamineMelatoninIon suppressionSerum

Introduction

Plasma and serum have a complex matrix that contain dissolved proteins and other trace materials that can often cause ion suppression when using liquid chromatography–mass spectrometry methods. Some of the main substances that can cause ion suppression are usually ionic substances or components that can result in charge transfer processes in the gas phase or in the electrospray ionisation process [1]. Phospholipids have been shown to cause high amounts of matrix effects. Phospholipids are the main component of cell membranes and the most significant types found in plasma are glycerophosphocholines and phosphatidylcholine [2].

The method described here is an LC–MS/MS method developed for the analysis of serotonin along with dopamine and melatonin. Serotonin (5-Hydroxytryptamine, 5-HT) is a neurotransmitter in the central and peripheral nervous systems of the body. It is involved in regulating blood pressure, smooth muscle contraction, pain sensation, it controls many brain functions and has been associated with sleep disruption and aggression. It has also been implicated in several pathological conditions, such as hypertension, migraine, depression, schizophrenia, anxiety, anorexia nervosa, dementia, and carcinoid syndrome [39]. Abnormal concentrations in plasma have been shown to reflect the serotonergic function in the central nervous system [4]. Serotonin levels, synthesis, uptake sites and receptor binding are all higher in the developing brain compared with adult values, and levels decline before puberty. This developmental process has been reported to be disrupted in autistic children [8].

Melatonin (N-acetyl-5-methoxytryptamine) is formed from an alternative metabolic pathway of serotonin in the human pineal gland at night under normal environmental conditions [10]. Melatonin plays an important role in the regulation of the circadian sleep–wake cycle. The concentration of melatonin in blood is changeable with the circadian rhythm, low around 10 pg mL−1 in the daytime and high around 25–120 pg mL−1 at night [11].

Dopamine (4-(2-aminoethyl)benzene-1,2-diol) is a neurotransmitter with its own system of neurons. It has been shown that serotonin can influence the dopaminergic system [12]. A dramatic loss of dopamine (DA) in the substantia nigra is constantly observed in the post mortem brains of patients with Parkinson’s disease [13]. The dopaminergic system has been reported to affect cognition, motor function, brain-stimulation reward mechanisms, eating and drinking behaviours, sexual behaviour, neuroendocrine regulation and selective attention [14]. The chemical structure of the target analytes is shown as inserts in Fig. 5b.

A variety of sample preparation techniques are used for the clean-up of complex matrices such as plasma and serum. These include solid phase extraction (SPE) [9, 10, 15], protein precipitation [1618], liquid–liquid extraction [1921] and online SPE [17, 22]. Many of these methods involve the use of relatively large amounts of solvents, long preparation times and incomplete removal of matrix components that may interfere with the analyte signal. The method described herein is a two-step preparation technique that utilises a protein precipitation step with a HybridSPE-Precipitation cartridge [23] for a rapid, cost effective and robust clean-up protocol. These marketed cartridges are specifically designed for the removal of phospholipids, a significant source of matrix interference in blood samples. Pucci et al. [24] monitored glycerophosphocholines and lysophosphatidylcholines which represent 70 and 10 % of total plasma phospholipids compared with reported a drastic decrease in the presence of these phospholipids when using the HybridSPE-Precipitation cartridges compared with simple protein precipitation leading to reduced matrix effects during analysis. Ardjomand-Woelkart et al. [25] utilised the 96-well HybridSPE-Precipitation technique for quantitation of CYP450 substrates/metabolites in rat plasma. Recoveries ranged from 69.25 ± 10.68 to 102.38 ± 7.22 %. Jiang et al. [26] saw a considerable reduction in matrix effects when using HybidSPE-precipitaion over protein precipitation for the determination of carboplatin in human plasma. A matrix factor of ~1 was seen for HybridSPE in comparison to 0.01 for protein precipitation. Recoveries were still quite low at <35 %.

This is the first reported use of HybridSPE-Precipitation cartridges for the rapid and sensitive analysis of serotonin, dopamine and melatonin in serum samples.

Materials and Methods

Materials

Chemicals used in this study included formic acid purchased from Merck (Darmstadt, Germany), ammonium acetate and acetic acid were purchased from Sigma-Aldrich (Dublin). Hydrochloric acid and all HPLC grade solvents (methanol, water, and acetonitrile) were purchased from Labscan (Dublin, Ireland). Nitrogen gas was purchased from Irish Oxygen (Ireland). All standard materials including the Supelco HybridSPE-Precipitation cartridges were purchased from Sigma-Aldrich (Dublin, Ireland) and the deuterated compounds (serotonin-d4, dopamine-d4 and melatonin-d4) were purchased from CDN Isotopes (Quebec, Canada). Serum samples were obtained from the Irish Blood Transfusion Service (IBTS).

Standard Preparation

Standards (serotonin, dopamine, and melatonin) in solvent (water) were prepared from a stock mixture of all compounds (57, 65 and 43 μmol L−1 for serotonin, dopamine and melatonin) by serial dilutions. Deuterated internal standards (serotonin-d4 and melatonin-d4) were subsequently added with final internal standard concentrations of 1.11 and 0.85 μmol L−1, respectively.

Standards in serum matrix were prepared by spiking standards from the stock solution into 100 μL of 0.1 % bovine albumin in phosphate buffer solution (pH 7), vortexing and proceeding with sample extraction.

Sample Preparation

Sample preparation involved addition of internal standards (20 μL) into each serum sample (100 μL), the samples were vortexed followed by the addition of acetonitrile (300 μL) with 1 % formic acid, vortexed again and then centrifuged for 10 min (3,000×g). The supernatants (~400 μL each) were put through HybridSPE-Precipitation cartridges, which were then eluted with methanol containing 0.1 mM ammonium acetate (600 μL; Fig. 2-5), and then the samples were dried under nitrogen and reconstituted in water (100 μL). An aliquot (5 μL) was injected into the LC–MS/MS.

Study of the Stability of Serotonin in Serum

A whole blood sample (2 mL) was collected by venepuncture and separated into two 1 mL aliquots, designated sample #1 and sample #2. Sample #1 was left at room temperature for 6 h, then placed in a fridge (4 °C) overnight, then centrifuged at 3,000 rpm for 15 min and stored at 4 °C for 4 days. Sample #2 was centrifuged immediately and stored at −80 °C for 4 days. Aliquots of each sample were prepared as in “Materials and Methods” and analysed on each of the 4 days to monitor the stability of serotonin. The purpose of this stability study was to compare the IBTS storage procedure which is followed by the sample #1 procedure in comparison to freezing samples at −80 °C, following sample collection.

Liquid Chromatography–Multiple Tandem Mass Spectrometry (LC–MS/MS)

An LC–MS/MS method was developed for the quantification of serotonin, dopamine and melatonin in serum. Separation was performed on an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA, USA) coupled to an API 3000 (Applied Biosystems, Warrington, UK) triple quadrupole mass spectrometer equipped with a Turbo-assisted Ionspray (ESI) ionisation source (Sciex, Toronto, Canada) operated in positive mode for the detection of analytes. Chromatographic separation was obtained using gradient elution on a reverse phase Hypersil Gold aQ column, 3 μm, 150 × 2.1 mm (Thermo) at 20 °C at a flow rate of 200 μL min−1. A binary gradient used was composed of mobile phase A, 0.1 % formic acid in water and mobile phase B, 0.1 % formic acid in MeOH. The ramped gradient profile was as follows: (a) 0 min 90 % A; (b) 3 min 80 % A; (c) 3.01 min 20 % A; (d) 5.5 min 50 % A (e) 7 → 12 min 20 % A; (f) 12.5 → 20 min 90 % A. The injection volume was 5 μL. MS detection was carried out between 1 and 15 min of the chromatography, total run time for the analysis was 20 min; the dwell time of the method was 1.82 min. Preselected Q1/Q3 ion pairs are outlined in Table 1; these were used to allow simultaneous detection of the target compounds. Optimised values for the mass spectrometer along with observed ion ratios can be seen in Table 1. Other parameters were kept constant for all analytes, such as nebuliser gas 8, curtain gas 12, CAD gas 5, Ion Source Voltage 5,000 V, Ion Source Temp 370 °C. Once every 3 months, the instrument was calibrated following manufacturer standard operating procedures; however, daily controls and calibration checks were performed for the target compounds to ensure detector stability. No deviation outside ±2.5 % was observed during this investigation.
Table 1

Optimised LC–MS/MS parameters

 

Mass [M + H]+

DP (FP)

Q1/Q3 masses

Collision energy (%)

Ion ratio

SD

Serotonin

177

28 (93)

177/160

18

1

 

177/132

30

0.14

0.009

177/115

40

0.17

0.014

Serotonin-d4

181

26 (94)

181/164

18

1

 

181/136

35

0.12

0.012

181/118

40

0.08

0.008

Dopamine

154

25 (80)

154/137

15

1

 

154/119

25

0.19

0.012

154/91

35

0.47

0.012

Melatonin

233

30 (80)

233/174

23

1

 

233/159

40

0.36

0.010

233/131

50

0.22

0.012

Melatonin-d4

237

33 (80)

237/178

23

1

 

237/163

40

0.35

0.008

237/135

55

0.22

0.012

Collision energy, declustering potential (DP), and focusing potential (FP) for each precursor/product ion pair of all compounds. SD is the standard deviation of the ion ratio over the concentration range 20–500 ng mL−1

Ion Suppression Post-Column Infusion Assessment

Post-column infusion experiments were conducted to determine ion suppression/enhancement effects using the approach described by Choi et al. [27]. The setup used in these experiments involved the infusion of a standard during the analysis of a blank matrix solution. The standard mixture (2.8, 2.2 and 3.3 μmol L−1for serotonin, melatonin and dopamine, respectively), monitored by MRM, was introduced at 5 μL min−1 into the LC eluent. A blank matrix (LC grade water) was injected (5 μL) using the autosampler into the LC system. The response of the standard mixture was monitored continuously to produce a profile of the matrix effect. Any drop in the constant baseline signal (‘suppression of signal’) indicates presence of interfering matrix components [2830]. The process was repeated with samples of serum. Five different serum samples were tested to access any possibility for sample-specific ion suppression effects.

Validation

Validation of the method was carried out in compliance with the EC [31] and ICH [32] guidelines assessing parameters such as specificity, linearity, limits of detection and quantitation:, trueness and precision [33].

Results and Discussion

The presence of phospholipids in a sample matrix can cause significant matrix effects when analysing small molecules in blood samples. Phospholipids are often extracted with analytes of interest and are difficult to resolve when using mass spectrometry. The HybridSPE-Precipitation cartridge facilitates protein precipitation together with solid phase extraction (SPE) technology for the targeted removal of phospholipids (i.e., through binding of phospholipids to the cartridge stationary phase) from plasma and serum samples. The unbound compounds of interest are then collected at the samples load step. A further elution step may be applied to increase the recoveries of the target compounds. No wash step to remove matrix interferences is required in this procedure.

Ismaiel et al. [2] have investigated the effect of several biological extract injections on the elution time of glycerophosphocholines to determine the effect of repeated injections on analytical performance. They commented that “monitoring phospholipids may provide a means to ensure the avoidance of matrix effects in each individual sample and may provide a more practical tool for avoiding matrix effects than commonly used post extraction addition and post-column infusion”. In another study, Ismaiel et al. [34] confirmed that ion suppression and enhancement coincided with the elution profiles of phospholipids in the analysis of chlorpheniramine in plasma.

There are a number of LC-tandem MS method that have been reported for the analysis of serotonin, dopamine and melatonin. Hows et al. [35] developed an assay for the determination of dopamine, norepinephrine, serotonin and cocaine in brain microdialysates. Monaghan et al. [17] developed a method for the analysis of serotonin in platelet depleted plasma for use as a biomarker in conditions such as functional gastrointestinal disorders. Protein precipitation was used for sample preparation followed by a cation exchange on-line clean-up step with analysis by LC–MS/MS. Peterson et al. [9] determined serotonin levels in serum using capillary electrophoresis–TOF–MS. The method removed both interferences and concentrated analytes from the serum using SPE with Oasis MXC (mixed mode cation exchange) columns. Miller et al. [18] reported an UPLC–MS method for the analysis of HIAA in plasma as an alternative to urine analysis for the diagnosis of midgut carcinoid tumours. LC–MS has been used to study the metabolism of serotonin in rat skin and serum for analysis of hydroxytryptophan in negative ion mode LC–ESI–MS [36, 37]. Table 2 shows a list of previously reported analytical methods describing the sample preparation technique used along with the recoveries and sensitivity of each method. The method described here provides excellent levels of accuracy, precision and recoveries which match or supersede previous studies. This method delivers sensitivity well below the mean quantities reported for these analytes in serum.
Table 2

Previously reported analytical methods describing the sample preparation technique along with the recoveries and sensitivity of each method

Analyte

Matrix

Detection method

Sample preparation

Sensitivity

Recovery

References

Melatonin

Saliva

LC–MS/MS

SPE (Nexus)

LOD 0.005 nmol L−1

LOQ 0.013 nmol L−1

77–97 %

Eriksson et al. [10]

Hydroxytryptophola

Urine

LC–MS

SPE (Thermo Hypersil Keystone C18)

LOD 1.2 and 2.0 nmol L−1 for blood and urine

LOQ 6.4 nmol L−1

94–104 %

Stephanson et al. [15]

Hydroxyindole acetic acida

Urine

LC–MS/MS

On-line SPE (HySphere resin GP cartridges 10 × 2 mm)

LOD <0.10 μmol L−1

LOQ 0.13 μmol L−1

96.5–99.6 %

de Jong et al. [22]

Serotonin

Serum

LC–MS/MS

On-line SPE Phenomenex strong cation exchange (SCX), 4.0 mm × 3.0 mm,

LOD 1.5 nmol L−1

LOQ 5 nmol L−1

98 % mean (range 89.5–115.5 nmol L−1)

Monaghan et al. [17]

Serotonin

Plasma

CE–MS

SPE Oasis MCX 10 mL (30 mg),

LOD 150 nmol L−1

71.6–3.1 %

Peterson et al. [9]

Hydroxyindole acetic acida

Plasma

LC–MS/MS

Protein precipitation

LOD 5 nmol L−1

LOQ 15 nmol L−1

97–113 %

Miller et al. [18]

Serotonin, hydroxyindole acetic acida, homovanillic acidb

Plasma

LC-CD

SPE-SAX Oasis cartridges

LOD 0.05 ng mL−1

LOQ 0.1 ng mL−1

>89 %

Saracino et al. [39]

Melatonin

Serum

LC-Fluorescence

LC-18 and Carbograph

LOD 0.013 nmol L−1

88.9 %

Lagana et al. [40]

aMetabolite of serotonin

bMetabolite of dopamine

Determination of Serotonin Using LC–MS/MS

Multiple reaction monitoring (MRM) studies were carried out to investigate the fragmentation of analyte precursor ions into their characteristic product ions and to establish the optimum collision energies. The most abundant MRM ions were chosen for quantitation (Table 1) with other optimised MRM ion combinations used for unequivocal identification of the analytes.

Optimisation of Chromatography

This method was originally developed for eight compounds, dopamine, 5-hydroxytryptophan (5-HTP), serotonin (5HT), tryptophan, 5-hydroxytryptophol (HTOL), 5-hydroxyindole acetic acid (HIAA), N-acetyl-hydroxytryptamine (N-acetyl-5HT) and melatonin. A gradient method was developed to resolve these compounds from each other. This was particularly important as HTOL was seen to interfere with serotonin quantitation giving a co-eluting peak due to similar MRM transitions. Another requirement of the method was the need to ensure dopamine did not co-elute with the instrument void volume (~1.5 min) as many matrix components and sample salts elute in this chromatographic region and caused ion suppression of the dopamine signal.

Figure 1a shows the chromatographic profile when using a Synergi Polar-RP column, Phenomenex (150 × 4.6 mm, 5 μm), this column allowed for good separation, but peak shape and sensitivity of some analytes were poor. It was decided to use a Hypersil Gold aQ column, Thermo, (3 μm, 150 × 2.1 mm) column (Fig. 1b) as this was seen to provide better results using the high aqueous mobile phase (90 % water:10 % MeOH both spiked with 0.05 % formic acid) that is present at the start of this method. Peak shape slightly improved, but dopamine still co-eluted with the instrument void volume. Figure 1c shows the same column and gradient as in Fig. 1b, but with an increased concentration of mobile phase buffer to 0.1 % formic acid. This improved the retention of analytes to the column and allowed the dopamine peak to elution later in the run. Also significant increases in analyte intensity were noted which enhanced the sensitivity of the method. Whilst only three analytes (serotonin, dopamine and melatonin) were eventually validated in this methodology, this chromatographic separation illustrates the multi-component separation and application of the chosen column. The complete suite of analytes is currently being validated and will be published separately.
https://static-content.springer.com/image/art%3A10.1007%2Fs10337-012-2330-5/MediaObjects/10337_2012_2330_Fig1_HTML.gif
Fig. 1

Chromatographic separation of eight compounds, 1 dopamine (m/z: 154/137), 2 5-Hydroxytryptophan (5-HTP) (m/z: 221/204), 3 Serotonin (5-HT) (m/z: 177/160), 4 Tryptophan (m/z: 205/188), 5 5-hydroxytryptophol (HTOL) (m/z: 178/160), 6 5-Hydroxyindole acetic acid (HIAA) (m/z: 192/146), 7N-acetyl-hydroxytryptamine (N-acetyl-5HT) (m/z: 219/160), 8 melatonin (m/z: 233/174) using a Synergi Polar-RP, Phenomenex (150 × 4.6 mm, 5 μm) with mobile phase 0.05 % formic acid in H2O and ACN; b Hypersil Gold aQ column, (3 μm, 150 × 2.1 mm) with the same mobile phase as a and c Hypersil Gold aQ column, (3 μm, 150 × 2.1 mm) with mobile phase 0.1 % formic acid in H2O and ACN)

HybridSPE-Precipitation for Sample Preparation

HybridSPE-Precipitation is a simple, one-step sample separation technique which is designed to remove high levels of endogenous protein and phospholipid interferences from plasma prior to LC–MS/MS analysis. The stationary phase of the cartridge acts as a chemical filter that specifically targets phospholipids, the retention mechanism is based on a highly selective Lewis acid–base interaction between the zirconia ions bonded to the stationary phase and the phosphonate moiety consistent with all phospholipids [23]. These cartridges allow for a significant reduction in sample preparation. On completion of a protein precipitation step, the sample is applied directly to the cartridge and allowed to pass through, collected and (in most cases) can then be analysed directly. The resulting eluent solution was dried under nitrogen and reconstituted in distilled H2O. Figure 2 shows a bar chart showing the different conditions (load eluent) used to optimise the SPE method. Figure 2-1 involved the procedure where the sample was loaded onto the HybridSPE cartridge with methanol containing 1 % formic acid (300 μL) [sample protein precipitation solvent (SPPS)], this load-eluent solvent was then collected from the cartridge, Fig. 2-2 involved the procedure where the sample was loaded onto the HybridSPE cartridge with acetonitrile containing 1 % formic acid (300 μL) (SPPS), this load-eluent solvent was then collected from the cartridge, Fig. 2-3 involved the procedure where the sample was loaded onto the HybridSPE cartridge with acetonitrile containing 1 % formic acid (300 μL) with methanol loaded separately (400 μL) (SPPS), and the combined eluents were collected from the cartridge, Fig. 2-4 involved the procedure where the sample was loaded onto the HybridSPE cartridge with acetonitrile containing 1 % formic acid (300 μL) (SPPS) with methanol containing ammonium acetate (0.1 mM; 200 μL) loaded separately and the combined eluents were collected from the cartridge, Fig. 2-5 involved the procedure where the sample was loaded onto the HybridSPE cartridge with acetonitrile containing 1 % formic acid (300 μL) (SPPS) with methanol containing ammonium acetate (0.1 mM; 600 μL) loaded separately and the combined eluents were collected from the cartridge, and Fig. 2-6 involved the procedure where the sample was loaded onto the HybridSPE cartridge with acetonitrile containing 1 % formic acid (300 μL) (SPPS) with methanol containing ammonium acetate (0.1 mM; 800 μL) loaded separately and the combined eluents were collected from cartridge. It was found that protein precipitation followed by HybridSPE clean-up with methanol (Fig. 2-1) or acetonitrile (Fig. 2-2) did not provide good recoveries for the target analytes. In fact, it was found that protein precipitation with acetonitrile spiked with 1 % formic acid and a methanol wash containing 0.1 mM ammonium acetate improved analyte recovery (Fig. 2-4 to Fig. 2-6). Figure 2 illustrates that a combined load eluent of 600 μL provided the best recovery (Fig. 2-5). The % recovery was calculated here as response of standard (2.84, 3.26 and 2.15 μmol L−1 for serotonin, dopamine and melatonin) spiked into blank serum compared to response of pure standard of identical concentration in water.
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Fig. 2

Bar chart illustrating the various samples load steps (1–6) applied so as to determine the optimum solvent composition to achieve the highest percentage recoveries for serotonin, dopamine and melatonin from the HybridSPE-PPT cartridge. % Recovery was calculated using the response of three standards (2.84, 3.26 and 2.15 μmol L−1 for serotonin, dopamine and melatonin) spiked into blank serum in comparison to standards spiked into water. 1 Sample protein precipitation (PPT) with methanol containing 1 % formic acid (300 μL), load and collect eluent from cartridge, 2 sample PPT with acetonitrile containing 1 % formic acid (300 μL), load and collect eluent from cartridge, 3 sample PPT with acetonitrile containing 1 % formic acid (300 μL) with methanol loaded separately (400 μL), load and collect combined eluent from cartridge, 4 sample PPT with acetonitrile containing 1 % formic acid (300 μL) with methanol containing ammonium acetate (0.1 mM; 200 μL) loaded separately and combined eluent collected from cartridge, 5 sample PPT with acetonitrile containing 1 % formaic acid (300 μL) with methanol containing ammonium acetate (0.1 mM; 600 μL) loaded separately and combined eluent collected from cartridge, and, 6 sample PPT with acetonitrile containing 1 % formaic acid (300 μL) with methanol containing ammonium acetate (0.1 mM; 800 μL) loaded separately and combined eluent collected from cartridge

Comparing this HybridSPE-PPT method with a developed C18 SPE method, there was a significant reduction in sample preparation time as well as solvent use. Figure 3 shows a flow chart comparing the procedure involved with the two afore-mentioned methods along with the time taken for each step and the solvent use of each technique. Sample preparation time is reduced by 70 min using the HybridSPE-PPT cartridge. The HybridSPE-PPT method consumed much less solvents, only requiring 1 mL of solvent for every sample compared to 12.8 mL using the C18 method. This is a significant handling and cost improvement, especially for routine clinical laboratories who deals daily with large numbers of samples.
https://static-content.springer.com/image/art%3A10.1007%2Fs10337-012-2330-5/MediaObjects/10337_2012_2330_Fig3_HTML.gif
Fig. 3

Flow chart comparing procedure, time required and solvent used for the elution of target compounds form a single serum sample, using a an Isolute C18 SPE method, and b the developed HybridSPE-PPT method. For serotonin both clean-up steps gave similar recoveries (averaging 98 and 95 %, respectively), however, as the flow chart outlines there is significant differences between the quantity of solvent consumed and processing time for both procedures

Validation

Matrix Effects

A post-column infusion experiment was carried out using the established method to investigate for matrix effects. The serum samples (See Online Resource 1) show large areas of ion suppression and enhancement, this is due to the matrix components that exit the column during the high organic wash stage of the gradient (9–13 min). By having a high aqueous mobile phase composition at the beginning of the gradient, the dopamine peak was separated from the area of ion suppression at 2 min but signal response of dopamine was still compromised by an ion suppression effect of ~20 % (Online resource 1b). Serotonin was affected to a lesser degree by ion suppression of ~5 %.

A dilution study was carried out on the serum sample to investigate the extent of ion suppression on the signal response of serotonin; a similar study was described by Villagrasa et al. [38]. Online resource 2 shows the effect of dilution on the signal response. On dilution, the response of the sample should match the theoretical response, but a slight increase was observed. This illustrates the impact of ion suppression on the serotonin signal response: as the matrix is diluted the components responsible for ion suppression are also diluted and, therefore, contribute less to signal suppression, resulting in a higher sample response. Although this proves that serotonin is affected by ion suppression the extent of ion suppression in this dilution study is relatively low at 5–8 %.

Recovery experiments were also carried out, where known concentrations of standards were spiked into serum matrices post extraction. The results can be seen in Table 3. Although the serum samples are affected by ion suppression, serotonin and melatonin still provide excellent recoveries (93–98 %). Even though dopamine does not have a deuterated internal standard for this study, it gave excellent quantitation using matrix matched standards. At the concentration of 653 nmol L−1 dopamine gave recoveries of 102 %. However, dopamine gave reduced recovery (65 %) at the low concentration standard (131 nmol L−1) and future experimentation will work on improving the quantitation of dopamine in clinical samples through the application of deuterated standards.
Table 3

Percentage recovery for compounds in serum after HybridSPE-Precipitation cartridge clean-up

 

Spiked concentration (nmol L−1)

Measured concentration (nmol L−1)

Recovery %

% RSD (n = 5)

Serotonin

284

268.9

94.78

2.77

1,135

1,093.9

96.38

3.32

Dopamine

131

84.8

64.97

9.31

653

664.4

101.77

6.51

Melatonin

215

201.4

93.54

7.35

861

850.4

98.76

1.34

Specificity

Retention time stability of an analyte is an important parameter to investigate during validation, as shifting retention times can lead to inaccuracies in identification as well as variations in the signal response of the analyte. The high selectivity of MRMs in LC–MS/MS allows for a higher degree of confidence in using retention time as identification marker. The EC council directive 2002/657/EC (implementing the 96/23) [31] suggests that retention time should not vary by more than 5 %. For this method, retention times were recorded over 3 days with three repeat injections per day of seven standards in the range of 0.086–3.286 μmol L−1 spiked into blank serum. Retention time shifts of ≤2.6, 3.6 and 1.2 % for serotonin, dopamine and melatonin, respectively, were observed. Analytes with an internal standard showed almost no difference in relative retention time as the deuterated internal references matched the retention characteristics of the analytes.

The three most abundant MRM transitions were chosen for each compound. Each precursor/product ion profile allows positive identification of the analytes and the Q1/Q3 ion pairs corresponds to 5.5 identification points according to the EC directive 2002/657/EC (implementing the 96/23) [31]. The ion ratios for the MRM transitions of each target compound were monitored over the range of 0.043–3.29 μmol L−1. The ion ratios and their standard deviations over this range are provided in Table 1. The ion ratios seen here were observed to be common for all calibrations in matrix and in samples, indicating that they can be used for analyte confirmation.

Linearity

The linear range in serum was 0.057–2.84, 0.065–3.27 and 0.021–2.15 μmol L−1 for serotonin, dopamine and melatonin, respectively. Table 4 shows the linear range, equation of line and correlation coefficient for each compound in serum. This method showed good linearity with correlation coefficients ≥0.9983 for serum (n = 5). In the equation of the line presented for each analyte in Table 4, “x” refers to the concentration of the analyte for serotonin, melatonin and dopamine in nmol/L. The “y” refers to the ratio of peak area of analyte to peak area of internal standard for serotonin and melatonin and peak area alone for dopamine.
Table 4

Results for linear range, trueness and precision for serotonin, melatonin and dopamine in serum

Serum

Linear range/calibration curve

Trueness

Intra + interday precision

Equation of the line

Linear range (μmol L−1)

R2

LOD (nmol L−1)

LOQ (nmol L−1)

Theoretical concentration (nmol L−1)

Measured concentration (nmol L−1)

RE %

RSD % n = 5

Concentration (nmol L−1)

Intraday RSD % n = 6

Interday RSD  % n = 9

Serotonin

0.0014x + 0.0184

2.837–0.057

0.9993

21.0

63.0

2,837

2,818.4

0.3

3.2

2,837

3.6

4.3

 

 

   

 

1,135

1,200.1

5.7

1.4

1,135

1.1

3.3

 

 

   

 

284

245.2

13.6

4.1

568

6.8

2.8

 

 

   

 

 

  

 

284

1.2

3.6

 

 

   

 

 

  

 

113

5.5

4.2

 

 

 

 

 

 

 

 

 

 

57

4.2

3.2

Dopamine

985.82x − 13245

3.268–0.065

0.9984

23.5

70.5

1,306

1,385.4

6.1

8.7

3,268

8.3

4.7

 

     

653

707.6

8.4

6.4

1,306

8.9

5.7

 

     

131

103.5

20.7

8.1

653

6.5

5.3

 

 

   

 

 

  

 

131

9.3

10.1

 

 

   

 

 

  

 

65

22.0

18.1

Melatonin

0.0011x + 0.0332

2.153–0.021

0.9986

3.2

15.4

2,153

2,045.3

5.0

1.4

2,153

3.2

3.2

 

 

   

 

861

857.5

0.4

3.9

861

3.8

3.9

 

 

   

 

215

188.7

12.3

1.8

431

3.7

3.7

 

 

   

 

 

  

 

215

3.5

3.5

 

 

 

 

 

 

 

 

 

 

43

7.0

7.4

          

21

7.1

8.3

Limits of Detection and Quantitation

The LOD and LOQs for serotonin in serum samples are given in Table 4. The LOD and LOQ were calculated for each compound in urine. The S/N was established from the standard deviation of the Y-intercept in six low range calibrations which is then divided by the slope of the average calibration for the six calibrations. This value was then multiplied by 3.3 for the LOD and 10 for the LOQ.

Trueness and Precision

The trueness of this method was expressed as percentage relative error of three different concentrations spiked into serum (water and 1 % bovine albumin in phosphate buffer pH 7). Medium and high concentration gave excellent recoveries for all compounds with percentage relative error (% RE) values ranging from 0.3 to 8.4 % (see Table 4). However, analytes showed reduced accuracy at low concentrations with % RE values at 284 and 215 nmol L−1 of 13.6 and 12.3 % for serotonin and melatonin, respectively, and 20.7 % for dopamine (at 131 nmol L−1). This loss in recovery is possible due to ion suppression which corresponds to the signal suppression observed for the post-column infusion experiment, i.e. 5 % for serotonin and melatonin and 20 % for dopamine.

In serum, the % RSD values for intra-day and inter-day precision were excellent for both serotonin and melatonin (Table 4) with both compounds showing good values even at concentrations close to their LODs: ~4 and 3 % for serotonin and LOD ~7 and 8 % for melatonin for intra-day and inter-day studies. Dopamine gave higher % RSD at the LOD level (65 nmol L−1) of 22.0 and 18.1 %, while all other higher concentrations provided good levels of precision ranging between 4.5 and 10.5 %.

Sample Stability

The stability of the analytes in urine and serum was investigated to develop the most appropriate sampling protocol. To obtain whole blood samples from the Irish Blood Transfusion Service (IBTS), an ethical assessment of the study was carried out by the ethics committee of the organisation and approval was granted to analyse samples for serotonin and dopamine. However, on receipt of the samples, it was observed that there may have been analyte degradation during the time from IBTS sampling to delivery to our laboratory. We, therefore, set about replicating the IBTS sampling procedure and comparing this IBTS sample to a fresh blood sample that was centrifuged immediately and placed in a −80 °C freezer. These samples were analysed over four consecutive days. From Fig. 4, it can be seen that there was extensive degradation over the 1st day using the IBTS sampling procedure. On the other hand, the sample that was stored at −80 °C and was immediately centrifuged on sampling (not always practical in a clinical setting) showed no sample degradation.
https://static-content.springer.com/image/art%3A10.1007%2Fs10337-012-2330-5/MediaObjects/10337_2012_2330_Fig4_HTML.gif
Fig. 4

Response of serotonin in serum over 4 days with two different storage conditions. Sample 1 followed the IBTS storage conditions; after sampling the sample was left at room temperature for 6 h, then placed in a fridge at 4 °C overnight, then centrifuged at 3,000g for 15 min and stored at 4 °C for 3 days. Sample 2 was centrifuged at 3,000g immediately and stored at −80 °C for 4 days

Application of Method

This developed method was applied to the analysis of serum samples (n = 19; Table 5). Calibration standards, blanks and controls were analysed along with the samples in each sequence. Figure 5 shows a chromatogram of serotonin, melatonin and dopamine in standard spiked into blank serum and in serum samples. This methodology is now being applied to a clinical screening programme and data from these studies will be published separately.
Table 5

Results for serotonin and dopamine detected in clinical samples

Sample

Serum (nmol L−1)

Serotonin

Dopamine

1

199.00

77.99

2

344.70

117.09

3

103.93

81.58

4

467.19

102.27

5

519.81

134.51

6

429.73

115.65

7

390.06

81.32

8

541.55

154.74

9

321.26

63.70

10

178.91

87.39

11

87.01

55.61

12

134.35

80.02

13

274.15

226.93

14

390.51

36.55

15

172.32

99.20

16

554.66

184.11

17

246.40

149.20

18

156.49

63.05

19

133.73

57.50

https://static-content.springer.com/image/art%3A10.1007%2Fs10337-012-2330-5/MediaObjects/10337_2012_2330_Fig5_HTML.gif
Fig. 5

Chromatogram of target compounds, a standards following extraction protocol, dopamine (peak 1; 2.78 min; 1.3 μmol L−1), serotonin (peak 2; 4.63 min; 2.8 μmol L−1) and melatonin (peak 4; 11.45 min: 2.2 μmol L−1) and internal standards serotonin-d4 (peak 3; 4.63 min; 1.1 μmol L−1) and melatonin-d4 (peak 5; 11.45 min; 0.9 μmol L−1), b clinical serum sample following extraction protocol, dopamine (peak 1; 2.87 min; 0.155 μmol L−1), serotonin (peak 2; 4.69 min; 0.542 μmol L−1) and melatonin (peak 4; 11.49 min; 0.009 μmol L−1) with internal standards serotonin-d4 (peak 3; 4.69 min; 1.1 μmol L−1) and melatonin-d4 (peak 5; 11.49 min; 0.9 μmol L−1)

Conclusion

This study outlines a validated method for serum for the analysis of serotonin, dopamine, and melatonin in serum. The method shows excellent results for accuracy and precision at concentrations expected in samples. It has been demonstrated that matrix effects are present with serum samples. This results in a reduction in analyte signal response at low concentration levels, particularly with the analysis of dopamine as it elutes close to an area of high ion suppression. Serotonin and melatonin are affected to a lesser extent by ion suppression and the additional presence of an internal standard for both compounds helps correct for these effects. Additionally, the study provided insight into the optimum storage conditions for the afore-mentioned biological samples. Finally, this method presents a simple, fast and reproducible sample preparation protocol using the HybridSPE-Precipitation cartridge. While dopamine showed issues of poor recovery at low concentrations, all parameters showed excellent results at levels present in serum samples. This shows that effective removal of phospholipids results in a sufficiently clean sample and that the HybridSPE-Precipitation cartridge can be applied to routine testing.

Acknowledgments

We gratefully acknowledge funding from the Irish Research Council for Science, Engineering and Technology (IRCSET) funding M. Moriarty. The Council of Directors, Technological Sector Research-Strand III 2006 Grant Scheme, awarded to Dr. A. Furey is also acknowledged for funding the formation of the ‘Team Elucidate’ research group. The Higher Education Authority (Programme for Research in Third-Level Institutions, Cycle 4 (PRTLI IV) National Collaboration Programme on Environment and Climate Changes: Impacts and Responses is also acknowledged.

Supplementary material

10337_2012_2330_MOESM1_ESM.ppt (87 kb)
Online resource 1: Example of a post-column infusion experiment for the evaluation of ion suppression in serum matrix for serotonin (a) and dopamine (b). Shaded areas correspond to elution times of analytes. Analyte standards (2.84, 3.26 and 2.15 μmol L−1 for serotonin, dopamine and melatonin) were infused at a rate of 10 μL min−1. This experiment was repeat with different serum samples (n = 5) (PPT 87 kb)
10337_2012_2330_MOESM2_ESM.ppt (70 kb)
Online resource 2: Dilution study for the assessment of matrix ion suppression on the target compound, serotonin, comparing various dilutions of serum. With no ion suppression effects from serum matrices at zero dilution, a 1 in 2 dilution of the samples should given exactly a 50 % response. This study determined that a 1 in 5 dilution is necessary to eliminate completely the influence of matrix ion suppression effects (PPT 69 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2012