Research Article
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Quantification of ghrelin and des-acyl ghrelin in human plasma by using cubicselected reaction-monitoring LC‑MS
Background: Ghrelin is a peptide hormone generally measured in plasma by immunoassays. LC‑MS/MS was investigated as an alternative method in particular for the quantification of the two forms of the peptide with improved selectivity. Materials & methods: A LC‑MS assay using a cubic-selected reaction-monitoring (LC‑SRM3 /MS) mode was developed for the quantification of ghrelin and des-acyl ghrelin in human plasma. Results: The LC‑SRM3 /MS method was found to be linear from 50–75 to 2500 pg/ml for the ghrelins using a 0.5-ml plasma sample. The accuracies and precisions at LOQ for des-acyl ghrelin (50 pg/ml) and ghrelin (75 pg/ml) were found to be better than 91 and 2%, respectively. Blood and plasma stabilization was found to be essential for good assay performance. Conclusion: Compared to the LC‑SRM/MS method the addition of an additional MS step did significantly improve the selectivity and therefore the sensitivity. The LC‑SRM3 /MS method could be successfully applied for the quantification of ghrelin and des-acyl ghrelin in human plasma samples.
Ghrelin is a peptide hormone characterized by Kojima et al. in 1999, which is mainly produced in the stomach and shows a strong growth hormone (GH)-realizing activity [1] . Two forms of ghrelin have been observed: des-acyl ghrelin and acyl-ghrelin (ghrelin) with a N-octanoyl modification on the third serine residue triggered by ghrelin O-acyltransferase [2] . It has been reported that only the acylated form activates the GH secretagogue receptor [1] . Ghrelin acts as an orexigenic peptide increasing appetite and food intake [3–6] . Additionally, ghrelin has been described in human, rat and mouse to contribute in body weight regulation, gastro intestinal motility, pituitary hormone axis regulation, metabolism of carbohydrate and numerous functions of the heart, kidney, pancreas, adipose tissues and gonads [7,8] . This 28-amino-acid peptide is derived from the 117-amino-acid precursor, preproghrelin. Des-acyl ghrelin, primarily supposed to be inactive, can counteract the effect of ghrelin [8] . Des-acyl ghrelin shows different functions, such as cell proliferation modulation and, to a limited extent, adipogenesis [9–11] .
10.4155/BIO.14.108 © 2014 Future Science Ltd
Jonathan Sidibé1, Emmanuel Varesio1 & Gérard Hopfgartner*,1 1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211 Geneva, Switzerland *Author for correspondence: Tel.: +41 22 379 63 44 Fax: +41 22 379 33 32 [email protected]
The human plasma level of ghrelin is approximately equal to 500 pg/ml [12] and its level decreases about two- to three-times after meal ingestion [13,14] . Ghrelin measurement in biological matrices is commonly performed by immuno assays such as ELISA or radioimmunoassay. Because ghrelin comes from a precursor protein, which is observed in two forms (acylated and des-acylated), the development of a specific antibody able to distinguish ghrelin from des-acyl ghrelin and from preproghrelin is difficult. Indeed, differences in ghrelin measurements can be observed depending on the commercial ELISA kits employed [15,16] . Multiple studies have demonstrated the benefits of LC‑MS in tandem mode (LC‑MS/MS) for the quantification of peptides in biological matrices [17,18] . Rauh et al. [16] reported a LC‑selected reaction monitoring (SRM)/MS method for ghrelin quantification in human plasma using column switching after protein precipitation. In this study, they have developed a method for the simultaneous quantification of ghrelin and des-acyl ghrelin with a LLOQ of 77 and 373 pg/ml for ghrelin and
Bioanalysis (2014) 6(10), 1373–1383
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Research Article Sidibé, Varesio & Hopfgartner des-acyl ghrelin, respectively, using 1 ml of plasma. While most LC‑MS quantification are performed in the SRM mode, it has been demonstrated that triple quadrupole/linear ion trap mass spectrometers, which can take advantage of the MS/MS/MS (MS3) capability by producing second-generation fragment ions, are a practicable approach for improving selectivity and LOQ of quantitative assays [19–22] . The present work aims to describe the application of the cubic SRM (LC‑SRM3 /MS) technique for the quantification of ghrelin and des-acyl ghrelin in human plasma, down to the pg/ml range using a simple automated sample preparation protocol. Materials & method Reagents & materials
Des-acyl ghrelin and ghrelin from human and rat (Figure 1) were obtained from PolyPeptide Laboratories France SAS (Strasbourg, France). Formic acid (FA) and trifluoroacetic acid (TFA) were obtained from Merck (Merck KGaA, Darmstadt, Germany). Sodium citrate tribasic dihydrate, hydrochloric acid (HCl) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Phenylmethylsulfonyl fluoride (PMSF) was obtained from Roche (Roche Diagnostic GmbH, Mannheim, Germany), sulfosalicylic acid (SSA) was obtained from Fluka (FlukaChemie GmbH, Buchs, Switzerland). HPLC grade acetonitrile was obtained from VWR (VWR International BVBA, Leuven, Belgium). Distilled deionized water was obtained from a Milli-Q Gradient A10 instrument (Millipore, MA, USA). Bovine citrate plasma was obtained from a local slaughterhouse (Meinier, Switzerland) and prepared from whole blood by centrifugation for 30 min at 1200 × g and 10°C. Human citrate plasma was obtained from the Centre de Transfusion Sanguine (Geneva University Hospital, Geneva, Switzerland) from nonfasted healthy volunteers who gave informed consent and was prepared from blood by centrifugation for 30 min at 1200 × g and 10°C. Both bovine and human plasma were kept frozen at -20°C prior use.
collection tubes. Plasma citrate samples were stabilized with 0.1% HCl and 1 mM PMSF in order to prevent ghrelin degradation by protein esterases. Standard solutions of human ghrelin and des-acyl ghrelin at 500 μg/ml were prepared in 0.1% FA, 0.1% BSA in water and working solutions of 20, 50, 100, 150, 200, 300, 400 and 500 ng/ml were prepared by dilution to the appropriate concentrations with a solution of 0.1% FA and 0.1% BSA in water. Standard solutions of rat ghrelin and des-acyl ghrelin were prepared in the same conditions and were used as internal standards (ISTD). Both standard and working solutions were stored at -20°C before use. Calibration curves and QC samples were prepared by spiking the working solutions into stabilized plasma to obtain concentrations ranging from 50 to 2500 pg/ml. Partial protein precipitation was carried out on 500 μl of plasma by adding 250 μl of SSA solution (100 mg/ml). After 1 min homogenization on a MixMate mixer (VaudauxEppendorf, Schönenbuch, Switzerland) at 1500 rpm, samples were centrifuged at 16,000 × g for 30 min at 4°C. After centrifugation, 600 μl of the clear supernatant were withdrawn and diluted to 1.5 ml with 0.1% FA in water. Samples were directly placed in the auto sampler, cooled at 4°C for analysis or stored at -20°C prior analysis. Liquid chromatography
LC separation was achieved on a SIL-HTC HPLC system (Shimadzu Corporation, Reinach, Switzerland) using a column-switching setup. The trapping column was a POROS R2, 20 μm 2.1 mm ID × 30 mm (Applied Biosystems, CA, USA) and the analytical column was an XBridge C18, 3.5 μm, 2.1 mm ID × 100 mm (Waters, MA, USA). Eluent A consisted of 0.2% FA, 0.01% TFA in water and eluent B consisted of 0.2% FA, 0.01% TFA in acetonitrile. Extract (1.4 ml) loaded onto the trapping column in the loading phase constituted of 0.2% FA, 0.01% TFA in water at a flow rate of 1 ml/min for 3 min. Peptides were eluted by a linear gradient starting from 5% B to 60% B in 3 min with a flow rate set at 0.3 ml/min. Mass spectrometry
Sample preparation
Blood stabilization was performed by the addition of aprotinin at 250 Kallikrein inhibitor units in the Key term LC‑SRM3 /MS: An acquisition mode specific for triple quadruple linear ion traps (QqQLIT). The first fragmentation step is achieved by quadrupole collisioninduced dissociation in q2, while the second fragmentation step is performed by trap collision induced in Q3. The additional fragmentation step improves the selectivity, while Q3 trapping improves the sensitivity.
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The SRM/MS and SRM3/MS experiments were performed on a QTRAP 5500 hybrid triple quadrupole linear ion trap mass spectrometer (AB Sciex, Concord, ON, Canada) operating in positive electrospray ionization using a TurboV ion source. SRM transitions were monitored with Q1 and Q3 quadrupoles set at unit mass resolution. The SRM transitions parameters are detailed in Supplementary Table 1. For SRM3/MS experiments, a fixed fill time of 100 ms was used for the ion trap. The excitation time was set at 25 ms and the
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
Results & discussion MS detection
Peptides such as ghrelin are generally ionized in electrospray under several charge states depending on the mobile phase conditions. In acidic conditions (pH: 2.6) four charge states (z = 4–7) were detected, from [M+7H] 7+ to [M+4H]4+, with the highest signal intensity observed for the [M+7H] 7+ and [M+6H] 6+ protonated molecules. With a mobile phase at pH 5.2, ghrelin peptides were observed as their [M+6H] 6+ to [M+4H]4+ charged state and signal intensity was the highest for the [M+4H] 4+ protonated molecules (Supplementary Figure 1) . Moreover, the use of dimethyl sulfoxide (25 or 50% DMSO in 0.1% FA in water) as supercharging agent (to promote one charge state) did not affect the charge distribution on ghrelin peptides probably due to their smaller sizes compared with proteins. Beside the loss in sensitivity, the charge states distribution may change over the concentration range and with sample background. In the present work, the more acidic pH for the mobile phase was selected and, to improve linearity and sensitivity, summing of multiple charge states and their corresponding fragments was employed for the quantitative analysis [23,24] . For each precursor ion submitted to collision-induced dissociation, three fragment ions were selected based on their signal intensity (Supplementary Figures 2 & 3) . In total, 18 SRM transitions for both ghrelin and des-acyl ghrelin were recorded during SRM acquisition and 16 for SRM3 (Supplementary Table 1) . Sample preparation
For the calibration curves and QC samples, bovine plasma was spiked with standard stock solutions prepared in 0.1% BSA and 0.1% FA in water to avoid unspecific binding to the sample tube walls [25,26] . Calibration curves and QC samples were prepared from independent stock solutions. Standard stock solutions were stable for at least 30 days at -20°C (data not shown). Subsequently, a partial protein precipitation was performed with the addition of SSA at a precipitation agent/sample ratio of 2/1 (v/v). After centrifugation, this method removes the major part of proteins larger
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Acylated ghrelin (octanoylated form)
O
excitation energy was fixed at 0.2 V. The MS instrument was controlled by Analyst software v.1.5.1 (AB Sciex). Data processing was performed using PeakView software v.1.0 (AB Sciex). Quantification was performed with MultiQuant software v.2.1 (AB Sciex) using a linear regression model with a 1/x 2 weighting. The integration algorithm was MQ4 with a Gaussian smoothing of a half-width equal to 2.0 points.
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H–GSSFLSPEHQ–RVQQRKESKK–PPAKLQPR–OH Des-acyl ghrelin 1
10 11
H–GSSFLSPEHQ–RVQQRKESKK–PPAKLQPR-OH
(M+H)+ GSS*FLSPEHQRVQQRKESKKPPAKLQPR
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GSS FLSPEHQRVQQRKESKKPPAKLQPR
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GSS*FLSPEHQKAQQRKESKKPPAKLQPR
3314.7 Da
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3188.7 Da
GSS*FLSPEHQKLQ-RKEAKKPSGRLKPR
3218.6 Da
GSS FLSPEHQKLQ-RKEAKKPSGRLKPR
3091.6 Da
Human
Rat
Bovine
Figure 1. Ghrelin and des-acyl ghrelin. (A) Human ghrelin and des-acyl ghrelin structures and (B) peptide sequences alignment of human, rat and bovine species. Amino acid sequence differences are shaded. S*: Octanoylated serine.
than 60 kDa according to SDS-PAGE observations A complete protein precipitation method could not be used to avoid co-precipitation of the analytes. The use of SSA prevents an evaporation step from occuring, which can be critical for the assay, and calls for a column-switching approach to load the sample onto the LC system. However, SSA is an organic acid that can affect peptide retention on the POROS trapping material. Thus, samples were diluted with water to reduce the organic acid concentration below 10% (v/v), which is suitable for adequate retention capability. Additionally, an autosampler stability test was performed to demonstrate the stability of the analytes in the autosampler conditions. Analytes were found to be stable at 4°C for 24 h (Supplementary Figure 5) . (Supplementary Figure 4) .
Column-switching LC setup A column-switching perfusion chromatography platform, allowing high loading solvent flow rate
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Key term Summed charge state: Electrospray ionization peptides are ionized in several charge states. Summed charge states consist of the summing of the signal from several cubic-selected reaction-monitoring transitions from different charge states.
(e.g., 1 ml/min) for fast injection of large sample volumes (e.g., 1.4 ml) without back pressure increase [27] was implemented. The POROS column made of poly(styrene-divinylbenzene), demonstrated strong robustness across multiple injections of partially precipitated plasma without clogging compared with C18 trapping cartridge packed with smaller particles (e.g., 3–5 μm particles), which was used initially. LC‑SRM3 /MS versus LC‑SRM/MS for plasma samples
After partial protein precipitation, the supernatant was injected onto a column-switching LC platform prior to the SRM or SRM 3 detection (Figure 2) . Despite the best signal intensity observed for the precursor ion at [M+7H] 7+, it could not be used to build SRM and SRM3 transitions due to lack of selectivity. In order to maximize signal-to-noise, we applied summed charge state, only SRM transitions showing sufficient selectivity were summed.
Thus, for the human des-acyl ghrelin, only three transitions from the same precursor ion ([M+5H]5+), could be used and summed. For human ghrelin, four transitions from two different charge states ([M+4H] 4+ and [M+5H]5+) could be used and summed (Supplementary Figures 3 & 4) . Representative chromatograms of the human ghrelin and desacyl ghrelin standards of 10 ng on-column are illustrated in Figure 3A . The retention times for human des-acyl ghrelin and ghrelin were 4.95 and 5.45 min, respectively. In this assay, summing ghrelin peptides SRM transition charge states improved the quantification performances in terms of sensitivity, accuracy and reproducibility. However, the LC‑SRM/MS method showed a lack in selectivity at low concentrations in blank bovine plasma, spiked bovine plasma and human plasma (Figure 3B–D) . Despite this, the assay could be validated and the selectivity under these conditions were found to not be sufficient to have a robust assay. One approach to improve selectivity of peptide analysis, focusing on sample preparation is immunoaffinity enrichment. Several studies have described validated biomarker assays using stable isotope standards capture by antipeptide antibodies [28–31] . In the present work, selectivity was improved on the MS
Loading Trapping column Pump 2 Injector Triple quadrupole linear ion trap (QqQ/LIT) Waste Pump 1 Elution
Pump 2
or
Analytical column Q1
q2
Q3/LIT
Waste Pump 1 Figure 2. Analytical workflow devised for the quantification of ghrelin and des-acyl ghrelin. Samples are loaded onto the trapping cartridge and eluted in back-flush mode onto the analytical column for peptide analysis by (A) LC‑selected reaction monitoring/MS or (B) LC‑cubic selected reaction monitoring/MS.
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
A 2.2e6
E 3.8e7
Des-acyl ghrelin Ghrelin
Intensity (cps)
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Intensity (cps)
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Figure 3. Representative chromatograms of summed traces for ghrelin and des-acyl ghrelin. (A & E) Analyzed in standard solution (10 ng on-column), (B & F) blank bovine plasma, (C & G) bovine plasma spiked at the LOQ and (D & H) human plasma by (A–D) LC‑selected reaction monitoring (SRM)/MS and (E–H) LC‑SRM3 /MS. The following transition used for SRM for des-acyl ghrelin were: sum of m/z 649.8 → 689.1, m/z 649.8 → 717.4 and m/z 649.8 → 667.3; and for ghrelin: sum of m/z 675.1 → 453.9, m/z 843.6 → 453.9, m/z 843.6 → 809.3 and m/z 843.6 → 513.3. The following transitions were used for SRM3 for des-acyl ghrelin: m/z 649.8 → 717.4 → sum of m/z 513.3 + 712.4 + 809.3 + 906.4 and m/z 812.0 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3; and for ghrelin: m/z 675.1 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3 and m/z 843.6 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3.
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Research Article Sidibé, Varesio & Hopfgartner side by including a second fragmentation step in the MS/MS dimension yielding to a LC‑MS3 method based on a triple quadrupole linear ion trap. In this mode, precursor ions are selected in Q1, then fragmented in q2 and product ions are collected in the linear ion trap to be further fragmented using resonance excitation. Ion chromatograms are then reconstructed from the selected second-generation fragment ions. For the LC‑SRM3 /MS acquisition, multiple secondgeneration fragment ions from the [M+4H] 4+ and [M+5H]5+ precursor ions were summed to generate the SRM3 ion chromatograms according to MS3 spectra shown in Supplementary Figures 5 & 6. Representative chromatograms of the LC‑SRM3 /MS acquisition mode are shown in Figure 3E for a standard solution of 10 ng on-column. The ion trap fill time was set to 100 ms to accumulate sufficient ions in the trap to gain sensitivity compared with the SRM/MS mode. Although the trap fill time of 100 ms results in a longer MS cycle time than the SRM/MS mode,
it was short enough to warrant the acquisition of at least ten data points across the chromatographic peak. The total analysis time was of 7.5 min. Compared with the LC‑SRM/MS mode, no interferences were detected in blank bovine plasma, spiked bovine plasma and human plasma (Figure 3F–H) . For the quantification performance, LOQ of the assay has been improved using LC‑SRM3 /MS compared with LC‑SRM/MS. For human ghrelin, the LOQ was at 75 pg/ml and for the des-acyl ghrelin at 50 pg/ml. Knowing that the normal human plasma level of ghrelin is approximately equal to 500 pg/ml [12] , the LC‑SRM 3 /MS method allows the quantification of human ghrelin with sufficient sensitivity and selectivity. Assay validation
For the LC‑SRM/MS method, the calibration curves were constructed using a linear regression model with a 1/x 2 weighting factor. The assay dynamic range
Table 1. Inter-assay precision (% residual standard deviation of the mean) and accuracy (%) based on quality control samples for ghrelin and des-acyl ghrelin measured by LC‑selected reaction monitoring/MS and cubic LC‑selected reaction monitoring/MS. Analyte
Concentration (pg/ml)
Measured concentration (pg/ml) Day 1
Day 2
n = 1
n = 2
n = 3
n = 4
n = 5
Average (pg/ml)
RSD (%)
Accuracy (%)
SRM ghrelin LOQ QC
100
91.94
106.9
114.8
89.14
107.8
102.1
11
97.9
Low QC
250
269.0
292.9
271.3
275.3
224.0
266.5
9.6
93.8
Medium QC
1500
1442
1572
1471
1464
1420
1474
4.0
102
High QC
2500
2143
2640
2395
2321
2550
2410
8.1
104
SRM des-acyl ghrelin LOQ QC
100
105.6
96.78
94.06
91.68
104.3
98.48
6
101.5
Low QC
250
216.8
257.5
217.5
246.6
220.6
231.8
8
107.9
Medium QC
1500
1428
1445
1498
1352
1625
1470
7
102.1
High QC
2500
2663
2195
2631
2461
2183
2427
9
103.0
SRM3 ghrelin LOQ QC
75
67.86
69.46
69.30
67.52
66.89
68.21
2
90.9
Low QC
100
111.1
110.9
117.1
116.1
111.8
113.4
3
113
Medium QC
500
498.0
450.0
465.1
464.8
420.8
459.7
6
91.9
High QC
2000
1801
1848
1754
1741
1796
1788
2
89.4
47.86
46.57
46.93
45.79
45.23
46.48
2
93.0
SRM3 des-acyl ghrelin LOQ QC
50
Low QC
100
112.1
111.4
112.9
115.2
115.8
113.5
2
113
Medium QC
500
544.5
502.1
503.3
531.7
521.2
520.6
4
104
High QC
2000
1818
2057
1791
1980
2148
1959
8
97.9
QC: Quality control; RSD: Residual standard deviation of the mean; SRM: Selected reaction monitoring.
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
6 5
Area ratio
was from 100 to 2500 pg/ml for des-acyl ghrelin (r2 = 0.992) and ghrelin (r2 = 0.995). The acceptance criterions for each back-calculated calibration standards were 15% deviation from the nominal value and 20% for the LLOQ over the five replicates. For intra-assay precision, the acceptance criterion were set to be lower than 15% for the calibration point standards and 20% at the LLOQ. The assay validation was performed over 2 days. Three replicates were analyzed on day 1 and two replicates analyzed on day 2. Most guidelines recommend five independent measurements to define interassay accuracy and precision. There are several accepted procedures to perform this type of investigation. To do it on two different days is one of them and accepted by GLP agencies. The intra-assay precision corresponding to the residual standard deviation of the mean and accuracy of the QC samples are presented in Table 1. The precision of the QC samples at the LOQ (LOQ QC) was 11% for the ghrelin and 6% for the des-acyl ghrelin. The accuracy of the LOQ QC samples was 97.7% for the ghrelin and 101.5% for the des-acyl ghrelin. Beside the assay could be validated the selectivity of the assay was not found to be sufficient to perform routine analysis at low concentration. For the LC‑SRM3 /MS method, the calibration curves were constructed using a linear regression model with a 1/x 2 weighting factor. The assay dynamic range was from 50 to 2500 pg/ml for the des-acyl ghrelin (r2 =0.9903) and from 75 to 2500 pg/ml for the ghrelin (r2 =0.9907). The same acceptance criterions described for the LC‑SRM/MS method were used for the LC‑SRM3 /MS method.
Research Article
Ghrelin Des-acyl ghrelin Ghrelin in stabilized plasma
4 3 2 1 0 0
30
60
90 120 Time (min)
150
180
Figure 4. Bench stability study of ghrelin in plasma (n = 5). Ghrelin (diamonds) and des-acyl ghrelin (squares) were periodically measured by LC‑selected reaction monitoring/MS in citrate plasma at room temperature over 3 h. Ghrelin was then measured in citrate plasma stabilized by the addition of HCl 0.5% (v/v) and phenylmethylsulfonyl fluoride at a final concentration of 1 mmol/l (triangles).
The precision for the LOQ QC samples was 2% for the des-acyl ghrelin and the ghrelin. The accuracy of the LOQ based on QC samples was 93.0% for the des-acyl ghrelin and 90.9% for the ghrelin (Table 1) . Compared with the method described by Rauh et al., no autosampler carry over was observed with the LC setup employed. Moreover, the developed method did not use solid-phase extraction in the sample preparation and used half of plasma sample volume (i.e., 500 μl compared with 1000 μl in Rauh et al.’s study).
Table 2. Intra-assay precision (% residual standard deviation of the mean) and accuracy (%) based on human plasma quality control samples for ghrelin and des-acyl ghrelin measured by cubic LC‑selected reaction monitoring/MS. Analyte
Concentration (pg/ml)
Measured concentration(pg/ml) n = 1
n = 2
n = 3
n = 4
n = 5
Average RSD (%) (pg/ml)
Accuracy (%)
SRM3 ghrelin Low QC
100
115.1
110.5
103.2
106.4
102.4
107.5
5
108
Medium QC
1500
1670
1681
1605
1645
1614
1643
2
110
High QC
2500
2293
2471
2377
2577
2504
2444
5
97.8
SRM3 des-acyl ghrelin Low QC
100
114.4
113.9
113.5
114.1
112.7
113.7
0.6
114
Medium QC
1500
1491
1504
1483
1489
1465
1486
1.0
99
High QC
2500
2443
2509
2529
2487
2465
2487
1.4
99.5
QC: Quality control; RSD: Residual standard deviation of the mean; SRM: Selected reaction monitoring.
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250 Des-acyl ghrelin
Ghrelin
Concentration (pg/ml)
200
150
100
50
0 Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Figure 5. Ghrelin and des-acyl ghrelin quantification in human plasma samples. Measurements were performed in triplicate (n = 3). Error bars represent one standard deviation.
Ghrelin & des-acyl ghrelin stability in plasma
Several studies described the instability of ghrelin in plasma [16,32] . Carboxylesterases were found to be mainly responsible of the ghrelin de-octanoylation, leading to the formation of des-acyl ghrelin. Bench stability of ghrelin and des-acyl ghrelin in human plasma was investigated over 3 h at room temperature. Without any plasma sample stabilization, more than 50% of the ghrelin is degraded in citrate plasma after 30 min (Figure 4) . The increase in des-acyl ghrelin detection across time incubation confirms that ghrelin is mainly degraded in des-acyl ghrelin in citrate plasma [32] . To inhibit degradation, PMSF at 1 mmol/l was used as esterases inhibitor in plasma samples. PMSF shows short half-lives times (35 min) in neutral conditions (pH 7–8), and can be improved under acidic conditions [33] . Thus, to ensure PMSF inhibition activity, plasma samples were acidified with 0.1% HCl 1 mol/l (v/v). With this stabilization method, ghrelin stability was preserved over 3 h at room temperature. In any case, to keep plasma samples on ice during the whole sample preparation procedure is recommended. Further investigations also confirmed that ghrelin and des-acyl ghrelin was found to be stable in human plasma after stabilization at -20°C for at least 7 days. Key term Surrogate matrix: As ghrelin and des-acyl ghrelin are endogenous analytes, bovine plasma was used as a surrogate matrix where the sequences in amino acids of the peptides are different.
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ISTD, surrogate matrix, recovery & matrix effects
In quantitative bioanalysis, ISTD are generally used to correct variability induced by the different steps of the analytical process. The ideal ISTD should demonstrate similar characteristics such as the analytes regarding extraction recovery, LC retention time and MS ionization efficiency. For that reason, stable isotopically labeled equivalents of the peptide are commonly used as ISTD for bioanalytical peptide LC–MS assays [18] . However, isotopically labeled peptides are not easily available, especially for peptides with modification on amino acid side chains, such as the ghrelin octanoyl moiety on the third serine residue. As a matter of fact, rat ghrelin and des-acyl ghrelin differ from human ghrelin and des-acyl ghrelin by only two amino acids giving a mass difference of 56.2 Da, and their physicochemical properties are similar to their human variants; for example, an isoelectric point of 10.56 versus 11.07 and a GRAVY index [34] . Therefore, rat ghrelin and des-acyl ghrelin were chosen as ISTD for this assay since their retention times are identical to the human peptides (i.e., des-acyl ghrelin is eluting at 4.95 min and ghrelin at 5.45 min) and they can be easily distinguished by mass spectrometry. As ghrelins are endogenous peptides, it is critical to use human plasma to prepare calibrations and QC samples and several alternative approaches have to be considered, such as the use of a surrogate matrix or use of labeled standards [35,36] . As already mentioned,
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
the amino acids sequences of ghrelins are speciesdependent and bovine plasma was selected as a surrogate matrix for human plasma. Recovery and matrix effects were monitored according Matuszewski et al. [37] and good correlations were found for human and bovine plasma. At 5 ng/ml, the recoveries (n = 5) were found to be 52.7% (coefficient of variation [CV]: 12.5%) for bovine plasma and 54.6% (CV: 12.1%) for human plasma, while the suppression effects were of 68.9% (CV: 2.3%) and 68.2% (CV: 5.2%) for bovine and human plasma, respectively. Matrix suppression was found to be significant but not relevant as an appropriate LOQ and assay performance could be achieved. Moreover, QC samples spiked in human plasma (previously left for 2 h at room temperature without a stabilization procedure to allow degradation of the ghrelins) were analyzed together with a calibration curve and QC samples prepared in bovine plasma. Table 2 shows the accuracy and precision results for human plasma QC at three levels of concentration over five replicates. All QC samples’ accuracy and precision was found to be below 15%, demonstrating good correlation for analytes solubility and extractability between bovine plasma and human plasma. Thus, bovine plasma is a good choice as a surrogate matrix because the risk of endogenous interferences is minimized and its composition is close to the authentic matrix (i.e., human plasma) [38] . Human samples LC‑SRM3 /MS measurements
The applicability of the assay was demonstrated for the analysis of des-acyl ghrelin and ghrelin in six healthy human volunteers. Each sample was measured in triplicate at different occasions using the LC‑SRM3 /MS method. The des-acyl ghrelin concentration ranged from 92.1 to 179.7 pg/ml and from 80.5 to 225.3 pg/ml for the ghrelin. For all six human samples analyzed, residual standard deviations of the mean of the calculated concentration were below 15% demonstrating good interassay performance on real samples (Figure 5) . In the six samples, concentrations of both peptides were lower than the typical
Research Article
concentration range measured by immunoassays [12] . Ghrelin levels decrease significantly after a meal and the lowest values have been recorded in fed samples [13,14] . In the present work, samples originated from nonfasted volunteers, so a low level of ghrelin are expected. Conclusion & future perspective The quantification of nontryptic peptides as a biomarker or as a pharmaceutical is of main importance. Selectivity in this field and a good limit of detection is therefore key and the LC‑SRM3 /MS approach is an interesting solution to improve selectivity and data quality without having to redevelop an assay. The sample preparation is quite generic and partial protein precipitation in combination with column-switching allows it to overcome carryover issues and to achieve good LOQs. Therefore, the present method could be extended to other peptides. Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future- science. com/doi/suppl/10.4155/BIO.14.108
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Methodology • A partial protein precipitation method combined with a column-switching LC analysis was developed for sample preparation and analytes separation. • Blood and plasma stabilization with aprotinin and an acidic solution of phenylmethylsulfonyl fluoride was found to be essential to stabilize the ghrelins. Bovine plasma was used as surrogate matrix for preparing calibration and quality control samples.
Conclusion • The present work presents a LC assay with mass spectrometric detection using cubic-selected reactionmonitoring (LC‑SRM3 /MS) mode to achieve quantification of ghrelin and des-acyl ghrelin in the pg/ml range with improved selectivity compared with the selected reaction-monitoring mode (LC‑SRM/MS).
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Research Article Sidibé, Varesio & Hopfgartner chromatography-tandem mass spectrometry in plasma, serum, and cell supernatants. Clin. Chem. 53(5), 902–910 (2007).
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Quantification of ghrelin and des-acyl ghrelin in human plasma by using cubicselected reaction-monitoring LC‑MS
Background: Ghrelin is a peptide hormone generally measured in plasma by immunoassays. LC‑MS/MS was investigated as an alternative method in particular for the quantification of the two forms of the peptide with improved selectivity. Materials & methods: A LC‑MS assay using a cubic-selected reaction-monitoring (LC‑SRM3 /MS) mode was developed for the quantification of ghrelin and des-acyl ghrelin in human plasma. Results: The LC‑SRM3 /MS method was found to be linear from 50–75 to 2500 pg/ml for the ghrelins using a 0.5-ml plasma sample. The accuracies and precisions at LOQ for des-acyl ghrelin (50 pg/ml) and ghrelin (75 pg/ml) were found to be better than 91 and 2%, respectively. Blood and plasma stabilization was found to be essential for good assay performance. Conclusion: Compared to the LC‑SRM/MS method the addition of an additional MS step did significantly improve the selectivity and therefore the sensitivity. The LC‑SRM3 /MS method could be successfully applied for the quantification of ghrelin and des-acyl ghrelin in human plasma samples.
Ghrelin is a peptide hormone characterized by Kojima et al. in 1999, which is mainly produced in the stomach and shows a strong growth hormone (GH)-realizing activity [1] . Two forms of ghrelin have been observed: des-acyl ghrelin and acyl-ghrelin (ghrelin) with a N-octanoyl modification on the third serine residue triggered by ghrelin O-acyltransferase [2] . It has been reported that only the acylated form activates the GH secretagogue receptor [1] . Ghrelin acts as an orexigenic peptide increasing appetite and food intake [3–6] . Additionally, ghrelin has been described in human, rat and mouse to contribute in body weight regulation, gastro intestinal motility, pituitary hormone axis regulation, metabolism of carbohydrate and numerous functions of the heart, kidney, pancreas, adipose tissues and gonads [7,8] . This 28-amino-acid peptide is derived from the 117-amino-acid precursor, preproghrelin. Des-acyl ghrelin, primarily supposed to be inactive, can counteract the effect of ghrelin [8] . Des-acyl ghrelin shows different functions, such as cell proliferation modulation and, to a limited extent, adipogenesis [9–11] .
10.4155/BIO.14.108 © 2014 Future Science Ltd
Jonathan Sidibé1, Emmanuel Varesio1 & Gérard Hopfgartner*,1 1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Life Sciences Mass Spectrometry, Quai Ernest-Ansermet 30, 1211 Geneva, Switzerland *Author for correspondence: Tel.: +41 22 379 63 44 Fax: +41 22 379 33 32 [email protected]
The human plasma level of ghrelin is approximately equal to 500 pg/ml [12] and its level decreases about two- to three-times after meal ingestion [13,14] . Ghrelin measurement in biological matrices is commonly performed by immuno assays such as ELISA or radioimmunoassay. Because ghrelin comes from a precursor protein, which is observed in two forms (acylated and des-acylated), the development of a specific antibody able to distinguish ghrelin from des-acyl ghrelin and from preproghrelin is difficult. Indeed, differences in ghrelin measurements can be observed depending on the commercial ELISA kits employed [15,16] . Multiple studies have demonstrated the benefits of LC‑MS in tandem mode (LC‑MS/MS) for the quantification of peptides in biological matrices [17,18] . Rauh et al. [16] reported a LC‑selected reaction monitoring (SRM)/MS method for ghrelin quantification in human plasma using column switching after protein precipitation. In this study, they have developed a method for the simultaneous quantification of ghrelin and des-acyl ghrelin with a LLOQ of 77 and 373 pg/ml for ghrelin and
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Research Article Sidibé, Varesio & Hopfgartner des-acyl ghrelin, respectively, using 1 ml of plasma. While most LC‑MS quantification are performed in the SRM mode, it has been demonstrated that triple quadrupole/linear ion trap mass spectrometers, which can take advantage of the MS/MS/MS (MS3) capability by producing second-generation fragment ions, are a practicable approach for improving selectivity and LOQ of quantitative assays [19–22] . The present work aims to describe the application of the cubic SRM (LC‑SRM3 /MS) technique for the quantification of ghrelin and des-acyl ghrelin in human plasma, down to the pg/ml range using a simple automated sample preparation protocol. Materials & method Reagents & materials
Des-acyl ghrelin and ghrelin from human and rat (Figure 1) were obtained from PolyPeptide Laboratories France SAS (Strasbourg, France). Formic acid (FA) and trifluoroacetic acid (TFA) were obtained from Merck (Merck KGaA, Darmstadt, Germany). Sodium citrate tribasic dihydrate, hydrochloric acid (HCl) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Phenylmethylsulfonyl fluoride (PMSF) was obtained from Roche (Roche Diagnostic GmbH, Mannheim, Germany), sulfosalicylic acid (SSA) was obtained from Fluka (FlukaChemie GmbH, Buchs, Switzerland). HPLC grade acetonitrile was obtained from VWR (VWR International BVBA, Leuven, Belgium). Distilled deionized water was obtained from a Milli-Q Gradient A10 instrument (Millipore, MA, USA). Bovine citrate plasma was obtained from a local slaughterhouse (Meinier, Switzerland) and prepared from whole blood by centrifugation for 30 min at 1200 × g and 10°C. Human citrate plasma was obtained from the Centre de Transfusion Sanguine (Geneva University Hospital, Geneva, Switzerland) from nonfasted healthy volunteers who gave informed consent and was prepared from blood by centrifugation for 30 min at 1200 × g and 10°C. Both bovine and human plasma were kept frozen at -20°C prior use.
collection tubes. Plasma citrate samples were stabilized with 0.1% HCl and 1 mM PMSF in order to prevent ghrelin degradation by protein esterases. Standard solutions of human ghrelin and des-acyl ghrelin at 500 μg/ml were prepared in 0.1% FA, 0.1% BSA in water and working solutions of 20, 50, 100, 150, 200, 300, 400 and 500 ng/ml were prepared by dilution to the appropriate concentrations with a solution of 0.1% FA and 0.1% BSA in water. Standard solutions of rat ghrelin and des-acyl ghrelin were prepared in the same conditions and were used as internal standards (ISTD). Both standard and working solutions were stored at -20°C before use. Calibration curves and QC samples were prepared by spiking the working solutions into stabilized plasma to obtain concentrations ranging from 50 to 2500 pg/ml. Partial protein precipitation was carried out on 500 μl of plasma by adding 250 μl of SSA solution (100 mg/ml). After 1 min homogenization on a MixMate mixer (VaudauxEppendorf, Schönenbuch, Switzerland) at 1500 rpm, samples were centrifuged at 16,000 × g for 30 min at 4°C. After centrifugation, 600 μl of the clear supernatant were withdrawn and diluted to 1.5 ml with 0.1% FA in water. Samples were directly placed in the auto sampler, cooled at 4°C for analysis or stored at -20°C prior analysis. Liquid chromatography
LC separation was achieved on a SIL-HTC HPLC system (Shimadzu Corporation, Reinach, Switzerland) using a column-switching setup. The trapping column was a POROS R2, 20 μm 2.1 mm ID × 30 mm (Applied Biosystems, CA, USA) and the analytical column was an XBridge C18, 3.5 μm, 2.1 mm ID × 100 mm (Waters, MA, USA). Eluent A consisted of 0.2% FA, 0.01% TFA in water and eluent B consisted of 0.2% FA, 0.01% TFA in acetonitrile. Extract (1.4 ml) loaded onto the trapping column in the loading phase constituted of 0.2% FA, 0.01% TFA in water at a flow rate of 1 ml/min for 3 min. Peptides were eluted by a linear gradient starting from 5% B to 60% B in 3 min with a flow rate set at 0.3 ml/min. Mass spectrometry
Sample preparation
Blood stabilization was performed by the addition of aprotinin at 250 Kallikrein inhibitor units in the Key term LC‑SRM3 /MS: An acquisition mode specific for triple quadruple linear ion traps (QqQLIT). The first fragmentation step is achieved by quadrupole collisioninduced dissociation in q2, while the second fragmentation step is performed by trap collision induced in Q3. The additional fragmentation step improves the selectivity, while Q3 trapping improves the sensitivity.
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The SRM/MS and SRM3/MS experiments were performed on a QTRAP 5500 hybrid triple quadrupole linear ion trap mass spectrometer (AB Sciex, Concord, ON, Canada) operating in positive electrospray ionization using a TurboV ion source. SRM transitions were monitored with Q1 and Q3 quadrupoles set at unit mass resolution. The SRM transitions parameters are detailed in Supplementary Table 1. For SRM3/MS experiments, a fixed fill time of 100 ms was used for the ion trap. The excitation time was set at 25 ms and the
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
Results & discussion MS detection
Peptides such as ghrelin are generally ionized in electrospray under several charge states depending on the mobile phase conditions. In acidic conditions (pH: 2.6) four charge states (z = 4–7) were detected, from [M+7H] 7+ to [M+4H]4+, with the highest signal intensity observed for the [M+7H] 7+ and [M+6H] 6+ protonated molecules. With a mobile phase at pH 5.2, ghrelin peptides were observed as their [M+6H] 6+ to [M+4H]4+ charged state and signal intensity was the highest for the [M+4H] 4+ protonated molecules (Supplementary Figure 1) . Moreover, the use of dimethyl sulfoxide (25 or 50% DMSO in 0.1% FA in water) as supercharging agent (to promote one charge state) did not affect the charge distribution on ghrelin peptides probably due to their smaller sizes compared with proteins. Beside the loss in sensitivity, the charge states distribution may change over the concentration range and with sample background. In the present work, the more acidic pH for the mobile phase was selected and, to improve linearity and sensitivity, summing of multiple charge states and their corresponding fragments was employed for the quantitative analysis [23,24] . For each precursor ion submitted to collision-induced dissociation, three fragment ions were selected based on their signal intensity (Supplementary Figures 2 & 3) . In total, 18 SRM transitions for both ghrelin and des-acyl ghrelin were recorded during SRM acquisition and 16 for SRM3 (Supplementary Table 1) . Sample preparation
For the calibration curves and QC samples, bovine plasma was spiked with standard stock solutions prepared in 0.1% BSA and 0.1% FA in water to avoid unspecific binding to the sample tube walls [25,26] . Calibration curves and QC samples were prepared from independent stock solutions. Standard stock solutions were stable for at least 30 days at -20°C (data not shown). Subsequently, a partial protein precipitation was performed with the addition of SSA at a precipitation agent/sample ratio of 2/1 (v/v). After centrifugation, this method removes the major part of proteins larger
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Acylated ghrelin (octanoylated form)
O
excitation energy was fixed at 0.2 V. The MS instrument was controlled by Analyst software v.1.5.1 (AB Sciex). Data processing was performed using PeakView software v.1.0 (AB Sciex). Quantification was performed with MultiQuant software v.2.1 (AB Sciex) using a linear regression model with a 1/x 2 weighting. The integration algorithm was MQ4 with a Gaussian smoothing of a half-width equal to 2.0 points.
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Figure 1. Ghrelin and des-acyl ghrelin. (A) Human ghrelin and des-acyl ghrelin structures and (B) peptide sequences alignment of human, rat and bovine species. Amino acid sequence differences are shaded. S*: Octanoylated serine.
than 60 kDa according to SDS-PAGE observations A complete protein precipitation method could not be used to avoid co-precipitation of the analytes. The use of SSA prevents an evaporation step from occuring, which can be critical for the assay, and calls for a column-switching approach to load the sample onto the LC system. However, SSA is an organic acid that can affect peptide retention on the POROS trapping material. Thus, samples were diluted with water to reduce the organic acid concentration below 10% (v/v), which is suitable for adequate retention capability. Additionally, an autosampler stability test was performed to demonstrate the stability of the analytes in the autosampler conditions. Analytes were found to be stable at 4°C for 24 h (Supplementary Figure 5) . (Supplementary Figure 4) .
Column-switching LC setup A column-switching perfusion chromatography platform, allowing high loading solvent flow rate
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Key term Summed charge state: Electrospray ionization peptides are ionized in several charge states. Summed charge states consist of the summing of the signal from several cubic-selected reaction-monitoring transitions from different charge states.
(e.g., 1 ml/min) for fast injection of large sample volumes (e.g., 1.4 ml) without back pressure increase [27] was implemented. The POROS column made of poly(styrene-divinylbenzene), demonstrated strong robustness across multiple injections of partially precipitated plasma without clogging compared with C18 trapping cartridge packed with smaller particles (e.g., 3–5 μm particles), which was used initially. LC‑SRM3 /MS versus LC‑SRM/MS for plasma samples
After partial protein precipitation, the supernatant was injected onto a column-switching LC platform prior to the SRM or SRM 3 detection (Figure 2) . Despite the best signal intensity observed for the precursor ion at [M+7H] 7+, it could not be used to build SRM and SRM3 transitions due to lack of selectivity. In order to maximize signal-to-noise, we applied summed charge state, only SRM transitions showing sufficient selectivity were summed.
Thus, for the human des-acyl ghrelin, only three transitions from the same precursor ion ([M+5H]5+), could be used and summed. For human ghrelin, four transitions from two different charge states ([M+4H] 4+ and [M+5H]5+) could be used and summed (Supplementary Figures 3 & 4) . Representative chromatograms of the human ghrelin and desacyl ghrelin standards of 10 ng on-column are illustrated in Figure 3A . The retention times for human des-acyl ghrelin and ghrelin were 4.95 and 5.45 min, respectively. In this assay, summing ghrelin peptides SRM transition charge states improved the quantification performances in terms of sensitivity, accuracy and reproducibility. However, the LC‑SRM/MS method showed a lack in selectivity at low concentrations in blank bovine plasma, spiked bovine plasma and human plasma (Figure 3B–D) . Despite this, the assay could be validated and the selectivity under these conditions were found to not be sufficient to have a robust assay. One approach to improve selectivity of peptide analysis, focusing on sample preparation is immunoaffinity enrichment. Several studies have described validated biomarker assays using stable isotope standards capture by antipeptide antibodies [28–31] . In the present work, selectivity was improved on the MS
Loading Trapping column Pump 2 Injector Triple quadrupole linear ion trap (QqQ/LIT) Waste Pump 1 Elution
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Waste Pump 1 Figure 2. Analytical workflow devised for the quantification of ghrelin and des-acyl ghrelin. Samples are loaded onto the trapping cartridge and eluted in back-flush mode onto the analytical column for peptide analysis by (A) LC‑selected reaction monitoring/MS or (B) LC‑cubic selected reaction monitoring/MS.
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
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Ghrelin
6.0
Ghrelin
6.0e4 4.0e4 2.0e4
4.5
6.0 H
Ghrelin
5.5 Time (min)
G
C Intensity (cps)
3.0e7
6.0
B 600
5.0
Time (min)
Des-acyl ghrelin
5.5
6.0
Ghrelin
1.0e5
250
Intensity (cps)
Intensity (cps)
Ghrelin
6.0e6
2.0e5
D
Des-acyl ghrelin
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200 150 100
8.0e4 6.0e4 4.0e4 2.0e4
50 4.5
5.0
5.5 Time (min)
6.0
4.5
5.0
5.5
6.0
Time (min)
Figure 3. Representative chromatograms of summed traces for ghrelin and des-acyl ghrelin. (A & E) Analyzed in standard solution (10 ng on-column), (B & F) blank bovine plasma, (C & G) bovine plasma spiked at the LOQ and (D & H) human plasma by (A–D) LC‑selected reaction monitoring (SRM)/MS and (E–H) LC‑SRM3 /MS. The following transition used for SRM for des-acyl ghrelin were: sum of m/z 649.8 → 689.1, m/z 649.8 → 717.4 and m/z 649.8 → 667.3; and for ghrelin: sum of m/z 675.1 → 453.9, m/z 843.6 → 453.9, m/z 843.6 → 809.3 and m/z 843.6 → 513.3. The following transitions were used for SRM3 for des-acyl ghrelin: m/z 649.8 → 717.4 → sum of m/z 513.3 + 712.4 + 809.3 + 906.4 and m/z 812.0 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3; and for ghrelin: m/z 675.1 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3 and m/z 843.6 → 453.9 → sum of m/z 513.3 + 712.4 + 809.3.
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Research Article Sidibé, Varesio & Hopfgartner side by including a second fragmentation step in the MS/MS dimension yielding to a LC‑MS3 method based on a triple quadrupole linear ion trap. In this mode, precursor ions are selected in Q1, then fragmented in q2 and product ions are collected in the linear ion trap to be further fragmented using resonance excitation. Ion chromatograms are then reconstructed from the selected second-generation fragment ions. For the LC‑SRM3 /MS acquisition, multiple secondgeneration fragment ions from the [M+4H] 4+ and [M+5H]5+ precursor ions were summed to generate the SRM3 ion chromatograms according to MS3 spectra shown in Supplementary Figures 5 & 6. Representative chromatograms of the LC‑SRM3 /MS acquisition mode are shown in Figure 3E for a standard solution of 10 ng on-column. The ion trap fill time was set to 100 ms to accumulate sufficient ions in the trap to gain sensitivity compared with the SRM/MS mode. Although the trap fill time of 100 ms results in a longer MS cycle time than the SRM/MS mode,
it was short enough to warrant the acquisition of at least ten data points across the chromatographic peak. The total analysis time was of 7.5 min. Compared with the LC‑SRM/MS mode, no interferences were detected in blank bovine plasma, spiked bovine plasma and human plasma (Figure 3F–H) . For the quantification performance, LOQ of the assay has been improved using LC‑SRM3 /MS compared with LC‑SRM/MS. For human ghrelin, the LOQ was at 75 pg/ml and for the des-acyl ghrelin at 50 pg/ml. Knowing that the normal human plasma level of ghrelin is approximately equal to 500 pg/ml [12] , the LC‑SRM 3 /MS method allows the quantification of human ghrelin with sufficient sensitivity and selectivity. Assay validation
For the LC‑SRM/MS method, the calibration curves were constructed using a linear regression model with a 1/x 2 weighting factor. The assay dynamic range
Table 1. Inter-assay precision (% residual standard deviation of the mean) and accuracy (%) based on quality control samples for ghrelin and des-acyl ghrelin measured by LC‑selected reaction monitoring/MS and cubic LC‑selected reaction monitoring/MS. Analyte
Concentration (pg/ml)
Measured concentration (pg/ml) Day 1
Day 2
n = 1
n = 2
n = 3
n = 4
n = 5
Average (pg/ml)
RSD (%)
Accuracy (%)
SRM ghrelin LOQ QC
100
91.94
106.9
114.8
89.14
107.8
102.1
11
97.9
Low QC
250
269.0
292.9
271.3
275.3
224.0
266.5
9.6
93.8
Medium QC
1500
1442
1572
1471
1464
1420
1474
4.0
102
High QC
2500
2143
2640
2395
2321
2550
2410
8.1
104
SRM des-acyl ghrelin LOQ QC
100
105.6
96.78
94.06
91.68
104.3
98.48
6
101.5
Low QC
250
216.8
257.5
217.5
246.6
220.6
231.8
8
107.9
Medium QC
1500
1428
1445
1498
1352
1625
1470
7
102.1
High QC
2500
2663
2195
2631
2461
2183
2427
9
103.0
SRM3 ghrelin LOQ QC
75
67.86
69.46
69.30
67.52
66.89
68.21
2
90.9
Low QC
100
111.1
110.9
117.1
116.1
111.8
113.4
3
113
Medium QC
500
498.0
450.0
465.1
464.8
420.8
459.7
6
91.9
High QC
2000
1801
1848
1754
1741
1796
1788
2
89.4
47.86
46.57
46.93
45.79
45.23
46.48
2
93.0
SRM3 des-acyl ghrelin LOQ QC
50
Low QC
100
112.1
111.4
112.9
115.2
115.8
113.5
2
113
Medium QC
500
544.5
502.1
503.3
531.7
521.2
520.6
4
104
High QC
2000
1818
2057
1791
1980
2148
1959
8
97.9
QC: Quality control; RSD: Residual standard deviation of the mean; SRM: Selected reaction monitoring.
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
6 5
Area ratio
was from 100 to 2500 pg/ml for des-acyl ghrelin (r2 = 0.992) and ghrelin (r2 = 0.995). The acceptance criterions for each back-calculated calibration standards were 15% deviation from the nominal value and 20% for the LLOQ over the five replicates. For intra-assay precision, the acceptance criterion were set to be lower than 15% for the calibration point standards and 20% at the LLOQ. The assay validation was performed over 2 days. Three replicates were analyzed on day 1 and two replicates analyzed on day 2. Most guidelines recommend five independent measurements to define interassay accuracy and precision. There are several accepted procedures to perform this type of investigation. To do it on two different days is one of them and accepted by GLP agencies. The intra-assay precision corresponding to the residual standard deviation of the mean and accuracy of the QC samples are presented in Table 1. The precision of the QC samples at the LOQ (LOQ QC) was 11% for the ghrelin and 6% for the des-acyl ghrelin. The accuracy of the LOQ QC samples was 97.7% for the ghrelin and 101.5% for the des-acyl ghrelin. Beside the assay could be validated the selectivity of the assay was not found to be sufficient to perform routine analysis at low concentration. For the LC‑SRM3 /MS method, the calibration curves were constructed using a linear regression model with a 1/x 2 weighting factor. The assay dynamic range was from 50 to 2500 pg/ml for the des-acyl ghrelin (r2 =0.9903) and from 75 to 2500 pg/ml for the ghrelin (r2 =0.9907). The same acceptance criterions described for the LC‑SRM/MS method were used for the LC‑SRM3 /MS method.
Research Article
Ghrelin Des-acyl ghrelin Ghrelin in stabilized plasma
4 3 2 1 0 0
30
60
90 120 Time (min)
150
180
Figure 4. Bench stability study of ghrelin in plasma (n = 5). Ghrelin (diamonds) and des-acyl ghrelin (squares) were periodically measured by LC‑selected reaction monitoring/MS in citrate plasma at room temperature over 3 h. Ghrelin was then measured in citrate plasma stabilized by the addition of HCl 0.5% (v/v) and phenylmethylsulfonyl fluoride at a final concentration of 1 mmol/l (triangles).
The precision for the LOQ QC samples was 2% for the des-acyl ghrelin and the ghrelin. The accuracy of the LOQ based on QC samples was 93.0% for the des-acyl ghrelin and 90.9% for the ghrelin (Table 1) . Compared with the method described by Rauh et al., no autosampler carry over was observed with the LC setup employed. Moreover, the developed method did not use solid-phase extraction in the sample preparation and used half of plasma sample volume (i.e., 500 μl compared with 1000 μl in Rauh et al.’s study).
Table 2. Intra-assay precision (% residual standard deviation of the mean) and accuracy (%) based on human plasma quality control samples for ghrelin and des-acyl ghrelin measured by cubic LC‑selected reaction monitoring/MS. Analyte
Concentration (pg/ml)
Measured concentration(pg/ml) n = 1
n = 2
n = 3
n = 4
n = 5
Average RSD (%) (pg/ml)
Accuracy (%)
SRM3 ghrelin Low QC
100
115.1
110.5
103.2
106.4
102.4
107.5
5
108
Medium QC
1500
1670
1681
1605
1645
1614
1643
2
110
High QC
2500
2293
2471
2377
2577
2504
2444
5
97.8
SRM3 des-acyl ghrelin Low QC
100
114.4
113.9
113.5
114.1
112.7
113.7
0.6
114
Medium QC
1500
1491
1504
1483
1489
1465
1486
1.0
99
High QC
2500
2443
2509
2529
2487
2465
2487
1.4
99.5
QC: Quality control; RSD: Residual standard deviation of the mean; SRM: Selected reaction monitoring.
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Research Article Sidibé, Varesio & Hopfgartner
250 Des-acyl ghrelin
Ghrelin
Concentration (pg/ml)
200
150
100
50
0 Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Figure 5. Ghrelin and des-acyl ghrelin quantification in human plasma samples. Measurements were performed in triplicate (n = 3). Error bars represent one standard deviation.
Ghrelin & des-acyl ghrelin stability in plasma
Several studies described the instability of ghrelin in plasma [16,32] . Carboxylesterases were found to be mainly responsible of the ghrelin de-octanoylation, leading to the formation of des-acyl ghrelin. Bench stability of ghrelin and des-acyl ghrelin in human plasma was investigated over 3 h at room temperature. Without any plasma sample stabilization, more than 50% of the ghrelin is degraded in citrate plasma after 30 min (Figure 4) . The increase in des-acyl ghrelin detection across time incubation confirms that ghrelin is mainly degraded in des-acyl ghrelin in citrate plasma [32] . To inhibit degradation, PMSF at 1 mmol/l was used as esterases inhibitor in plasma samples. PMSF shows short half-lives times (35 min) in neutral conditions (pH 7–8), and can be improved under acidic conditions [33] . Thus, to ensure PMSF inhibition activity, plasma samples were acidified with 0.1% HCl 1 mol/l (v/v). With this stabilization method, ghrelin stability was preserved over 3 h at room temperature. In any case, to keep plasma samples on ice during the whole sample preparation procedure is recommended. Further investigations also confirmed that ghrelin and des-acyl ghrelin was found to be stable in human plasma after stabilization at -20°C for at least 7 days. Key term Surrogate matrix: As ghrelin and des-acyl ghrelin are endogenous analytes, bovine plasma was used as a surrogate matrix where the sequences in amino acids of the peptides are different.
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ISTD, surrogate matrix, recovery & matrix effects
In quantitative bioanalysis, ISTD are generally used to correct variability induced by the different steps of the analytical process. The ideal ISTD should demonstrate similar characteristics such as the analytes regarding extraction recovery, LC retention time and MS ionization efficiency. For that reason, stable isotopically labeled equivalents of the peptide are commonly used as ISTD for bioanalytical peptide LC–MS assays [18] . However, isotopically labeled peptides are not easily available, especially for peptides with modification on amino acid side chains, such as the ghrelin octanoyl moiety on the third serine residue. As a matter of fact, rat ghrelin and des-acyl ghrelin differ from human ghrelin and des-acyl ghrelin by only two amino acids giving a mass difference of 56.2 Da, and their physicochemical properties are similar to their human variants; for example, an isoelectric point of 10.56 versus 11.07 and a GRAVY index [34] . Therefore, rat ghrelin and des-acyl ghrelin were chosen as ISTD for this assay since their retention times are identical to the human peptides (i.e., des-acyl ghrelin is eluting at 4.95 min and ghrelin at 5.45 min) and they can be easily distinguished by mass spectrometry. As ghrelins are endogenous peptides, it is critical to use human plasma to prepare calibrations and QC samples and several alternative approaches have to be considered, such as the use of a surrogate matrix or use of labeled standards [35,36] . As already mentioned,
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Quantification of ghrelin & des-acyl ghrelin in human plasma by using cubic-selected reaction-monitoring LC‑MS
the amino acids sequences of ghrelins are speciesdependent and bovine plasma was selected as a surrogate matrix for human plasma. Recovery and matrix effects were monitored according Matuszewski et al. [37] and good correlations were found for human and bovine plasma. At 5 ng/ml, the recoveries (n = 5) were found to be 52.7% (coefficient of variation [CV]: 12.5%) for bovine plasma and 54.6% (CV: 12.1%) for human plasma, while the suppression effects were of 68.9% (CV: 2.3%) and 68.2% (CV: 5.2%) for bovine and human plasma, respectively. Matrix suppression was found to be significant but not relevant as an appropriate LOQ and assay performance could be achieved. Moreover, QC samples spiked in human plasma (previously left for 2 h at room temperature without a stabilization procedure to allow degradation of the ghrelins) were analyzed together with a calibration curve and QC samples prepared in bovine plasma. Table 2 shows the accuracy and precision results for human plasma QC at three levels of concentration over five replicates. All QC samples’ accuracy and precision was found to be below 15%, demonstrating good correlation for analytes solubility and extractability between bovine plasma and human plasma. Thus, bovine plasma is a good choice as a surrogate matrix because the risk of endogenous interferences is minimized and its composition is close to the authentic matrix (i.e., human plasma) [38] . Human samples LC‑SRM3 /MS measurements
The applicability of the assay was demonstrated for the analysis of des-acyl ghrelin and ghrelin in six healthy human volunteers. Each sample was measured in triplicate at different occasions using the LC‑SRM3 /MS method. The des-acyl ghrelin concentration ranged from 92.1 to 179.7 pg/ml and from 80.5 to 225.3 pg/ml for the ghrelin. For all six human samples analyzed, residual standard deviations of the mean of the calculated concentration were below 15% demonstrating good interassay performance on real samples (Figure 5) . In the six samples, concentrations of both peptides were lower than the typical
Research Article
concentration range measured by immunoassays [12] . Ghrelin levels decrease significantly after a meal and the lowest values have been recorded in fed samples [13,14] . In the present work, samples originated from nonfasted volunteers, so a low level of ghrelin are expected. Conclusion & future perspective The quantification of nontryptic peptides as a biomarker or as a pharmaceutical is of main importance. Selectivity in this field and a good limit of detection is therefore key and the LC‑SRM3 /MS approach is an interesting solution to improve selectivity and data quality without having to redevelop an assay. The sample preparation is quite generic and partial protein precipitation in combination with column-switching allows it to overcome carryover issues and to achieve good LOQs. Therefore, the present method could be extended to other peptides. Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future- science. com/doi/suppl/10.4155/BIO.14.108
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Methodology • A partial protein precipitation method combined with a column-switching LC analysis was developed for sample preparation and analytes separation. • Blood and plasma stabilization with aprotinin and an acidic solution of phenylmethylsulfonyl fluoride was found to be essential to stabilize the ghrelins. Bovine plasma was used as surrogate matrix for preparing calibration and quality control samples.
Conclusion • The present work presents a LC assay with mass spectrometric detection using cubic-selected reactionmonitoring (LC‑SRM3 /MS) mode to achieve quantification of ghrelin and des-acyl ghrelin in the pg/ml range with improved selectivity compared with the selected reaction-monitoring mode (LC‑SRM/MS).
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Research Article Sidibé, Varesio & Hopfgartner chromatography-tandem mass spectrometry in plasma, serum, and cell supernatants. Clin. Chem. 53(5), 902–910 (2007).
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