Anal Bioanal Chem (2015) 407:497–507 DOI 10.1007/s00216-014-8214-9
RESEARCH PAPER
Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry Ugo Bussy & Huiyong Wang & Yu-Wen Chung-Davidson & Weiming Li
Received: 23 August 2014 / Revised: 19 September 2014 / Accepted: 22 September 2014 / Published online: 31 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultrahigh performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiplecharged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.
U. Bussy : H. Wang : Y. 200 Da Daughter ion with m/z < 200 Da
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0
Normalized signal on noise ratios (%)
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pr La ey m PQ pr La e R m y PQ Fa pr ey R La PQ Fa m -2 pr R Fa ey La PQ -RP R 1 m F pr ey a-R La P2 P m XR pr Fa ey PX -1a La R m F apr 1b La ey G m n pr R e H La y I G m n pr ey RH II G nR H III
Normalized intensities (%)
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Fig. 3 Effect of daughter ion weight on MRM of peptides. Comparison between small fragments (m/z200 Da) on signal intensity (A) and signal-tonoise ratio (B) normalized to the higher signal
freshly prepared at 12 concentrations (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng/mL) for each validation day. Targetlynx function of Masslynx software was used for sample processing through 1/χ least-square regression of the relative response of standard area vs. internal standard area(s). The matrix effect parameters were assessed by using the signal areas in five samples made with extracted plasma, five blank plasma and five samples made in standard solution. Extraction recovery parameters were calculated by the area ratio of five plasma samples spiked pre-extraction over the
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La m La pre y m PQ pr La e R m Fa pr y P Q e y R La PQ Fa m pr R Fa 2 ey La PQ -RP m R Fa 1 pr La ey P -RP m pr XR 2 Fa ey La PXR 1a m pr Fa1b La ey G m nR pr H La ey I G m n pr ey RH II G nR H III
and processed for calibration and quantification of the analytes with TargetLynx software from Waters (Fig. 3).
Simultaneous determination of GnIH and GnRH
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peptides. Protein precipitation and SPE condition were not modified compared to the recently reported method for simultaneous quantification of GnRHs [36]. However, as reported by Chambers et al. [34, 35] peptide analysis is challenging mainly due to their low stability in solution, adsorption phenomena, and their MS signal dilution into several charge states formed through atmospheric pressure ionization. Stability of analytes and internal standards was investigated in spiked plasma extract at -20 °C devoid of light. Concentrations were normalized vs. first measurement and monitored over 5 h. All analyte stabilities were greater than 80 %. Peptide stabilities strongly depend on the amino acid residues and their sequences. Also, GnRH I and PQRFa-2 signal strength tends to decrease when they are monitor over a day (data not shown). The stability of some peptides suggests that analysis should be carried out immediately after the extraction process to limit variation due to peptide degradation or adsorption (or both). Moreover, samples should be stored at −80 °C and thaw just before analysis to minimize the time spent in the autosampler. Nonetheless, since the analytes and the internal standards degraded in a similar fashion, the measurement can always be back-calculated accordingly and stay valid. The stabilities of GnRHs, GnIHs and related RFamide peptides, and ISs are summarized in Fig. 4. Significant variation were observed in standard solution and attributed to adsorption phenomena. Variations were minimized by the usage of low binding materials and by adding 0.05 % of rat plasma to the solution [34, 35, 38]. Three columns, BEH C18 (1.0×50 mm 1.7 μm particle size), BEH C18 (2.1×50 mm 1.7 μm particle size), and the recent CSH C18 (2.1×50 mm 1.7 μm particle size), were evaluated for their separation of the neuropeptides by liquid chromatography with a binary gradient between water (0.1 %
mean area of plasma samples spiked post-extraction rather than over pure standard solution to limit the contribution of the endogenous background. Matrix effect and extraction recovery parameters for plasma and brain extracts have been previously compared for GnRHs and similar results were observed [36]. Therefore, extensive method validation experiments were run only for the plasma matrix and similarity for the brain extract was assumed. The extraction recovery parameter was estimated by the ratio of the concentrations measured in extracted spiked plasma to extracted plasma spiked post-extraction after endogenous background subtraction. Matrix effect parameter (ME) was evaluated through the ratio of peak area post-extraction after blank subtraction to the peak area in standard solution as described by Matuszewki et al. [37]. The accuracy and precision were validated in five replicates of three concentrations over three consecutive days. Intraday parameters were estimated by the comparison of five replicates analyzed in the same day while interday parameters were based on the data acquired for five replicates over the three validation days. Precision was determined by the coefficient of variation and accuracy by the ratio of the measured concentration to that of spiked standard. All values are expressed with standard deviation range.
Results and discussion Sample preparation and LC separation
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Fig. 4 Stability of GnRH and GnIH and related RFamide peptides and internal standards in plasma extract spiked at the concentration of 5 ng/mL for five replicates
Relative intensity (%)
Sample preparation was developed to satisfy the quantitative analyses of all lamprey GnRHs and GnIH and related
90
80 Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2 Lamprey PXRFa-1a Lamprey PXRFa-1b
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80 Lamprey GnRH-I Lamprey GnRH-II Lamprey GnRH-III Grass Puffer LP Hagfish RP1 LHRH
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Relative standard error (%) Time (hour) LFa1a LFa2 LRP1 LFa1b LRP2 GLP LFa HRP1 LHRH GnRH I GnRH II GnRH III 0 2.6 2.5 2.5 2.4 5.2 4.6 4.8 5.1 3.8 4.8 4.5 5.7 1 4.7 1.9 2.7 2.9 6.3 7.0 6.7 6.2 5.9 8.0 7.1 5.3 2 2.3 1.5 2.9 2.8 4.8 5.4 3.2 4.4 4.7 4.5 4.4 4.5 3 2.8 2.1 1.5 1.8 2.1 1.2 2.2 2.2 1.7 2.1 1.5 3.0 4 1.9 1.7 1.0 2.1 1.3 1.3 2.4 1.3 2.0 1.8 1.1 5.1 5 2.1 1.5 1.7 2.4 1.6 1.6 2.0 0.8 1.5 1.4 0.9 1.8
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Table 2 Summary of the MSMS parameters for the three GnIHs and three related RFamide peptides, three GnRHs and three internal standards (ISs)
GnIHs and related peptides
GnRHs
ISs
CV (V)
CE (V)
Parent m/z (Da)
Parent charge state
Daughter m/z (Da)
Daughter attribution
Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2
40 40 40 40
20 10 10 25
732.5 436.4 394.0 667.4
[M+3H]3+ [M+4H]4+ [M+3H]3+ [M+2H]2+
897.5 509.4 481.4 546.8
[Y5 +2H]2+ [Y11 +3H]3+ [Y7 +2H]2+ [Y4 +H]+
Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III LHRH Grass puffer LP Hagfish RP1
40 40 40 40 40 40 40 40
15 15 20 15 23 20 25 20
495.5 478.8 613.9 610.9 630.1 591.8 731.0 533.9
[M+3H]4+ [M+5H]5+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+3H]3+ [M+2H]2+
579.0 562.6 741.7 1048.9 773.5 748.0 888.8 848.6
[B12 +2H]2+ [Y20 +4H]4+ [B6 +H]+ [B8 +H]+ [B6 +H]+ [Y7 +H]+ [Y7 +H]+ [Y7 +H]+
formic acid) and acetonitrile. CSH phase is recommended for peptide analyses due to its positively charged Si-stationary phase showing better performances than the other two columns regarding to the peak shape and sensitivity. As mentioned by Chambers et al. [34, 35] each sample set was preceded by nine injections of rat-precipitated plasma to enhance peak shape and sensitivity. Liquid chromatography separation was achieved within 8.5 min, repeatability of retention times was insured by the comparison of spiked extracted plasma, and standard solution displayed in Fig. 1 (Table 2). MSMS parameters MSMS analysis is a challenging step during LC-MS/MS determination of peptides in biological samples. To address this issue, ionization and collision-induced dissociation (CID) condition were optimized for GnRH and GnIH and related RFamide peptide analyses with a Waters TQ-S triple quadrupole mass spectrometer. Instrument resolution was decreased to monitor multiple charged compounds. MRM parameters
were manually optimized with 10 μL injections of 1 μg/mL solutions. For each peptide, collision energy (CE) was optimized for the highest intensities of charge states. Although some studies have resulted in the successful use of low m/z product ions with good sensitivity [29–31], the selection of ions with higher m/z, especially in the case of multiple charged macro molecules such as peptides, tends to lead to better selectivity [32]. A single transition was selected for each peptide with respect to sensitivity and specificity. In fact, two types of transitions were investigated and split into two categories; i) the smallest m/z daughter ions (200 Da) produced under lower CID energies. Transitions exploiting low energies are summarized in Table 1 while transition exploiting higher energies were used as follows: parent m/z > daughter m/z (collision energy/ V); Lamprey PQRFa 732.5 >129.3 (50), Lamprey PQRFa-2 436.4>109.7 (40), Lamprey PQRFa-RP1394.0>119.9 (40), Lamprey PQRFa-RP2 667.4>119.8 (50), Lamprey PXRFa1a 495.5>119.8 (30), Lamprey PXRFa-1b 478.8>119.9 (30), Lamprey GnRH I 614.0>173.0 (45), Lamprey GnRH II
Table 3 General performances of the determination of GnRHs and GnIHs and related RFamide peptides by LC-MSMS in biological matrix
GnIHs and related peptides
GnRHs
Linear range
R2
LOD (ng/mL)
LOQ (ng/mL)
Retention time (min)
Lamprey PQRFa Lamprey PQRFa-2
0.1–50 0.025–50
0.9979 0.9983
0.1 0.05
0.5 0.1
5.41 2.48
Lamprey PQRFa-RP1 Lamprey PQRFa-RP2 Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III
0.01–50 0.075–50 0.025–50 0.05–50 0.01–50 0.01–50 0.250–50
0.9989 0.9978 0.9997 0.9978 0.9979 0.9985 0.9987
0.025 0.1 0.1 0.075 0.1 0.025 0.25
0.1 0.25 0.25 0.25 0.25 0.1 0.75
3.42 4.45 2.70 2.56 3.29 4.60 2.54
Simultaneous determination of GnIH and GnRH
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Table 4 Matrix effect and recovery parameters for GnIH and related RFamide, GnRH and internal standard peptides
GnIHs and related peptides
GnRHs
ISs
Matrix effect (%) [mean±SD]
Recovery (%) [mean±SD]
Selected IS(s)
Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2
98.8±4.0 91.4±6.3 84.7±6.0 98.3±4.0
81.7±4.9 79.6±3.9 64.1±4.9 79.7±4.9
GLP GLP HRP1 GLP
Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III LHRH Grass Puffer LP Hagfish RP1
75.5±2.6 70.3±1.3 94.9±3.4 91.3±5.1 100.3±11.2 99.0±3.6 94.8±4.8 76.1±4.3
90.1±6.7 104.5±7.3 88.2±4.3 72.9±2.3 74.3±5.7 80.7±3.3 85.5±6.1 68.3±12.7
GLP and HRP1 GLP and HRP1 LHRH LHRH LHRH GnRH’s IS GnIH’s IS GnIH’s IS
Table 5 Intra- and interday accuracy and precision of the determination of GnIHs, related RFamides, and GnRHs by LC-MSMS in biological matrix Intraday (n=5) Added (ng mL−1) GnIHs and related peptides
Lamprey PQRFa
Lamprey PQRFa-2
Lamprey PQRFa-RP1
Lamprey PQRFa-RP2
Lamprey PXRFa-1a
GnRHs
Lamprey PXRFa-1b
Lamprey GnRH I
Lamprey GnRH II Lamprey GnRH III
a
Accuracy=100[determined concentration/added]
c
Coefficient of variation
[mean±SD]a (ng mL−1)
Accuracyb (%)
Precisionc (%)
[mean±SD]a (ng mL−1)
Accuracyb (%)
Precisionc (%)
0.50
0.44±0.06
88.0
13.6
0.48±0.09
96.7
18.4
5.00
4.85±0.40
96.7
8.3
5.34±0.61
106.8
12.3
50.00
56.14±4.83
112.3
8.6
53.08±5.84
106.2
11.7
0.50
0.56±0.09
111.2
16.7
0.53±0.08
106.4
16.4
5.00
4.74±0.54
94.9
11.4
4.81±0.56
96.1
11.2
50.00
53.47±7.34
106.9
13.7
51.71±6.84
103.4
13.7
0.50
0.54±0.05
108.0
9.4
0.51±0.07
101.2
13.3
5.00
4.18±0.65
83.6
15.6
4.28±0.58
85.6
11.5
50.00
43.51±4.96
87.0
11.4
43.6±5.03
87.3
10.1
0.50
0.46±0.07
91.6
15.7
0.45±0.07
90.4
13.5
5.00
4.65±0.72
93.0
15.5
4.58±0.61
91.7
12.1
50.00
46.38±5.41
92.8
11.7
47.15±5.79
94.3
11.6
0.50
0.58±0.09
116.8
16.2
0.57±0.08
113.2
16.6
5.00
5.08±0.56
101.6
11.1
5.31±0.56
106.2
11.2
50.00
54.79±8.20
109.6
15.0
53.24±6.99
106.5
14.0
0.50
0.51±0.08
102.8
15.2
0.51±0.08
101.5
15.6
5.00
4.73±0.57
94.5
12.1
5.11±0.66
102.2
13.2
50.00
55.65±6.60
111.3
11.9
53.66±6.10
107.3
12.2
0.50
0.47±0.05
94.8
11.4
0.47±0.06
93.2
11.3
5.00
4.10±0.49
82.0
11.9
4.59±0.62
91.8
12.4
50.00
40.97±1.31
81.9
3.2
42.95±3.68
85.9
7.4
0.50
0.41±0.07
81.2
16.8
0.41±0.06
82.9
12.6
5.00
4.57±0.72
91.4
15.8
4.49±0.52
89.7
10.4
50.00
41.80±3.92
83.6
9.4
41.94±3.95
83.9
7.9
5.00
4.02±0.46
80.4
11.4
4.09±0.46
82.0
9.1
50.00
43.58±5.88
87.2
13.5
43.7±3.68
87.4
7.4
Average determined concentration±standard deviation
b
Interday (n=15)
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611.0>173.0 (35), and Lamprey GnRH III 420.0>110.0 (35). The performances were evaluated on five replicates made of a plasma extract spiked with 1 ng/mL of neuropeptides. Figure 2 highlights the approach for the selection of the MRM transition with the example of Lamprey PXRFa-1b. First of all, the full scan spectrum showed the signal dilution into several charge states; secondly, the two different types of collision-induced dissociations. The fragment at 119.9 Da obtained under collision energy of 30 V showed the best sensitivity. On the other hand, the fragment at 562.6 Da obtained under lower collision energy (15 V) was more specific to Lamprey PXRFa-1b. While sensitivity is a key parameter for quantitative analyses, determination methods need to be specific to minimize the noise amplitude. With respect to the quantitative analysis based on MSMS detection, a primary concern remains to be the signal-to-noise ratio (S/N). Therefore, S/N was measured for two types of transitions in plasma spiked with 1 ng/mL of neuropeptides. Experiments were carried out in plasma to allow for the best evaluation of the endogenous background on intensities and S/N. Intensities and S/N were normalized to the higher value for each peptide. As shown in Fig. 3, the best S/N was obtained for the transition at 478.8>562.6. Despite a sensitivity that was twice of the other transition, the transition at 478.8>119.9 showed a S/N five times lower. A similar trend was observed for other peptides (Fig. 3). Six of the nine investigated neuropeptides showed higher intensities for the transition using daughter ions with m/z under 200 Da, showing better sensitivities. Figure 3B shows normalized S/N for all nine peptides with two types of transitions. Contrary to the intensity, all nine peptides displayed higher S/N for the MSMS transition exploiting m/z greater than 200 Da. Opposite to the signal broadening on low-resolution instruments, the MSMS
General performances Limit of determination (LOD) and limit of quantification (LOQ) are defined as S/Ns of 3 and 10, respectively. LOD and LOQ were first evaluated in standard solution and then determined in spiked extracted plasma, and subsequently summarized in Table 2. Relative intensity to the selected internal standard(s) was plotted against the concentrations to build calibration curves from 0.01 to 50 ng/mL. Linearity was validated by the correlation coefficient of linear regression (R2) ranging from 0.9978 to 0.9997 while LOQ in plasma samples were systematically under 1 ng/mL, allowing for the detection of the targeted neuropeptides in brain and plasma matrices of sea lamprey (Table 3). Matrix effect and extraction recovery As expected for LC-MSMS analysis of molecules in biological samples, matrix effects were observed. Matrix effects were evaluated through comparison of peptide solutions made in standard solution and extracted plasma spiked postextraction at the final concentration of 5 ng/mL. Extraction recovery parameters were calculated for each analyte and internal standard based on the ratio of matrix spiked before and after extraction. For GnRHs, LHRH has already been
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Fig. 5 Average concentrations of neuropeptides in plasma (A) and brain tissues (B)
analysis of multiple-charged molecules allows for the selection of daughter ions with larger m/z than the parent ion m/z. Except for lamprey PQRFa-RP2, the selected transitions exploiting this last attribute were summarized in Table 1. The selection of specific MSMS transitions combined with liquid chromatography separation allows for the determination of the targeted peptides without interferences.
Simultaneous determination of GnIH and GnRH
demonstrated as a suitable internal standard. For GnIHs, Grass puffer LP, and Hagfish RP1 were evaluated by comparing matrix effects and extraction recovery parameters. Matrix effects and extraction recoveries determined for lamprey PQRFa, PQRFa-2, and PQRFa-RP2 were similar to those of grass puffer LP while the value determined for lamprey PQRFa-RP1 suggests hagfish RP1 as a more suitable internal standard (Table 1). Lamprey PXRFa-1a and PXRFa-1b showed dichotomous behaviors. Indeed, matrix effect values were similar to that of hagfish RP1 while extraction recovery values were closer to that of grass puffer LP. This justifies the usage of both hagfish RP1 and grass puffer LP as internal standards for lamprey PXRFa-1a and PXRFa-1b (Table 4).
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GnRH III concentration was measured at 1.02±0.50 ng/mL and 12.38±3.19 ng/mL in male and female plasma extracts, respectively. Lamprey PQRFa RP2 and PXRFa 1b were respectively measured at 1.77±0.18 ng/mL and 0.86±0.15 ng/ mL in the male brain and 2.79±0.77 ng/mg and 1.55±0.36 ng/ mL in the female brain. The significant difference between plasma and brain matrices and/or prespermiating male and preovulatory female observed for seven of the nine targeted neuropeptides confirmed the performance of the method and its applicability to biological study as routine analysis.
Conclusion Accuracy and precision Precision and accuracy were evaluated on the same day through analyses of five replicates of three concentrations. The validation run was repeated over three consecutive days. Intraday accuracy ranged from 80.4 to 116.8 % while interday accuracy ranged from 82.0 to 113.2 %, as shown in Table 3. Precision ranged from 3.2 to 16.8 % from intraday analyses and from 7.4 to 18.4 % from interday analyses (Table 5). As required by the FDA guidelines, precision was less than 20 % for the lowest validation concentration (LQC) while the two other concentrations (MQC and HQC) were above 15 %. Moreover, accuracy values do not exceed the limit of ±20 % of the expected value and fulfilled the requirement for biological sample analysis. Method reproducibility and automation is also dependent upon retention time repeatability which were evaluated for each peptide through five replicates at 5 ng/mL over three consecutive days (n=15). The inter day reproducibility of retention time was validated by the standard deviations ranging from 0.01 to 0.08 min with precision ranging from 0.52 to 2.35 % (coefficient of variation) (Table 5). Application The method was used to analyze GnRHs and GnIHs and related RFamide peptides in lamprey plasma and brain tissues. With respect to the expression of neuropeptides in brain, the determined concentrations were expressed relative to wet tissue mass, which ranged from 41.3 to 90.0 mg. Animals were grouped by sex and maturation corresponding to prespermiating males and preovulatory females. Large variations were expected as supported by interindividual variation and peptide stability. Five GnIHs and related RFamide peptides and two GnRHs were determined in biological matrix. Differences were observed between plasma and brain tissues (Fig. 5). Highest concentrations were measured for GnRH III in plasma while lamprey PQRFa-RP2 and PXRFa-1b were the most abundant in the brain. Lamprey
The full analytical approach for the determination of six GnIHs and related RFamide peptides and three GnRHs have been optimized, validated, and applied to biological samples. The study resulted in a rapid, precise, selective, and sensitive method for the determination of nine neuropeptides that play key roles in the gonadotropin regulation. This is the first study using SPE/LC-MSMS to quantify GnIH and related RFamide peptides. In addition, simultaneous determination of both GnIH and GnRH had never been reported. Matrix effect, recovery, and accuracy and precision parameters were evaluated while optimizing the selectivity and sensitivity combination in biological samples. This method was applied in analysis of sea lamprey brain tissue. Positive results are also expected on plasma matrix since they have shown higher levels of GnRHs [36, 39]. Analytical performances described here suggest that the method could be employed for routine analysis of biological samples. With slight modification, this methodology could be adapted to determine neuropeptides in other vertebrate species including humans. Acknowledgments The authors thank Professor Daniel Jones and Lijun Chen of the Michigan State University MS Facility for helpful advice. This study was funded by a grant from the Great Lakes Fishery Commission.
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U. Bussy et al. determination of leuprolide in human serum. J Chromatogr A 877: 3194–3200 21. Niwa M, Enomoto K, Yamashita K (1999) Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr B 729:245–253 22. Guzman NA (2000) Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry. J Chromatogr B 749:197–213 23. Holzgrabe U, Nap C-J, Almeling S (2011) Use of collision induced dissociation mass spectrometry as a rapid technique for the identification of pharmacologically active peptides in pharmacopoeial testing. J Pharm Biomed Anal 55:957–963 24. Kafka AP, Rades T, McDowell A (2010) Rapid and specific highperformance liquid chromatography for the in vitro quantification of d-Lys6–GnRH in a microemulsion-type formulation in the presence of peptide oxidation products. Biomed Chromatogr 24:132–139 25. Breci LA, Tabb DL, Yates JR, Wysocki VH (2003) Cleavage Nterminal to proline: analysis of a database of peptide tandem mass spectra. Anal Chem 75:1963–1971 26. Kicman AT, Parkin MC, Iles RK (2007) An introduction to mass spectrometry based proteomics—detection and characterization of gonadotropins and related molecules. Mol Cell Endocrinol 260– 262:212–227 27. Fricker LD, Lim J, Pan H, Che F-Y (2006) Peptidomics: identification and quantification of endogenous peptides in neuroendocrine tissues. Mass Spectrom Rev 25:327–344 28. van den Broek I, Sparidans RW, Schellens JHM, Beijnen JH (2008) Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry. J Chromatogr B 872:1–22 29. Hatziieremia S, Kostomitsopoulos N, Balafas V, Tamvakopoulos C (2007) A liquid chromatographic/tandem mass spectroscopic method for quantification of the cyclic peptide melanotan-II. Plasma and brain tissue concentrations following administration in mice. Rapid Commun Mass Spectrom 21:2431–2438 30. Cass RT, Villa JS, Karr DE, Schmidt DE (2001) Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns. Rapid Commun Mass Spectrom 15: 406–412 31. Kobayashi N, Kanai M, Seta K, K-i N (1995) Quantitative analysis of synthetic human calcitonin by liquid chromatography-mass spectrometry. J Chromatogr B 672:17–23 32. Chang D, Kolis SJ, Linderholm KH, Julian TF, Nachi R, Dzerk AM, Lin PP, Lee JW, Bansal SK (2005) Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC– MS/MS. J Pharm Biomed Anal 38:487–496 33. Piehowski PD, Petyuk VA, Orton DJ, Xie F, Moore RJ, RamirezRestrepo M, Engel A, Lieberman AP, Albin RL, Camp DG, Smith RD, Myers AJ (2013) Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis. J Proteome Res 12:2128–2137 34. Chambers EE, Legido-Quigley C, Smith N, Fountain KJ (2012) Development of a fast method for direct analysis of intact synthetic insulins in human plasma: the large peptide challenge. Bioanalysis 5: 65–81 35. Chambers EE, Lame ME, Bardsley J, Hannam S, Legido-Quigley C, Smith N, Fountain KJ, Collins E, Thomas E (2013) High sensitivity LC–MS/MS method for direct quantification of human parathyroid 1–34 (teriparatide) in human plasma. J Chromatogr B 938:96–104 36. Wang H, Chung-Davidson YW, Li W (2014) Identification and quantification of sea lamprey gonadotropin-releasing hormones by electrospray ionization tandem mass spectrometry. J Chromatogr A 1345:98–106
Simultaneous determination of GnIH and GnRH 37. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal Chem 75: 3019–3030 38. van Midwoud PM, Rieux L, Bischoff R, Verpoorte E, Niederländer HAG (2007) Improvement of recovery and repeatability in liquid
507 chromatography–mass spectrometry analysis of peptides. J Proteome Res 6:781–791 39. Chung-Davidson YW, Wang H, Siefkes MJ, Bryan MB, Wu H, Johnson NS, Li W (2013) Pheromonal bile acid 3ketopetromyzonol sulfate primes the neuroendocrine system in sea lamprey. BMC Neurosci 14:11
RESEARCH PAPER
Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry Ugo Bussy & Huiyong Wang & Yu-Wen Chung-Davidson & Weiming Li
Received: 23 August 2014 / Revised: 19 September 2014 / Accepted: 22 September 2014 / Published online: 31 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultrahigh performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiplecharged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.
U. Bussy : H. Wang : Y. 200 Da Daughter ion with m/z < 200 Da
100
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40
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0
Normalized signal on noise ratios (%)
B
pr La ey m PQ pr La e R m y PQ Fa pr ey R La PQ Fa m -2 pr R Fa ey La PQ -RP R 1 m F pr ey a-R La P2 P m XR pr Fa ey PX -1a La R m F apr 1b La ey G m n pr R e H La y I G m n pr ey RH II G nR H III
Normalized intensities (%)
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La m
Fig. 3 Effect of daughter ion weight on MRM of peptides. Comparison between small fragments (m/z200 Da) on signal intensity (A) and signal-tonoise ratio (B) normalized to the higher signal
freshly prepared at 12 concentrations (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng/mL) for each validation day. Targetlynx function of Masslynx software was used for sample processing through 1/χ least-square regression of the relative response of standard area vs. internal standard area(s). The matrix effect parameters were assessed by using the signal areas in five samples made with extracted plasma, five blank plasma and five samples made in standard solution. Extraction recovery parameters were calculated by the area ratio of five plasma samples spiked pre-extraction over the
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0
La m La pre y m PQ pr La e R m Fa pr y P Q e y R La PQ Fa m pr R Fa 2 ey La PQ -RP m R Fa 1 pr La ey P -RP m pr XR 2 Fa ey La PXR 1a m pr Fa1b La ey G m nR pr H La ey I G m n pr ey RH II G nR H III
and processed for calibration and quantification of the analytes with TargetLynx software from Waters (Fig. 3).
Simultaneous determination of GnIH and GnRH
501
peptides. Protein precipitation and SPE condition were not modified compared to the recently reported method for simultaneous quantification of GnRHs [36]. However, as reported by Chambers et al. [34, 35] peptide analysis is challenging mainly due to their low stability in solution, adsorption phenomena, and their MS signal dilution into several charge states formed through atmospheric pressure ionization. Stability of analytes and internal standards was investigated in spiked plasma extract at -20 °C devoid of light. Concentrations were normalized vs. first measurement and monitored over 5 h. All analyte stabilities were greater than 80 %. Peptide stabilities strongly depend on the amino acid residues and their sequences. Also, GnRH I and PQRFa-2 signal strength tends to decrease when they are monitor over a day (data not shown). The stability of some peptides suggests that analysis should be carried out immediately after the extraction process to limit variation due to peptide degradation or adsorption (or both). Moreover, samples should be stored at −80 °C and thaw just before analysis to minimize the time spent in the autosampler. Nonetheless, since the analytes and the internal standards degraded in a similar fashion, the measurement can always be back-calculated accordingly and stay valid. The stabilities of GnRHs, GnIHs and related RFamide peptides, and ISs are summarized in Fig. 4. Significant variation were observed in standard solution and attributed to adsorption phenomena. Variations were minimized by the usage of low binding materials and by adding 0.05 % of rat plasma to the solution [34, 35, 38]. Three columns, BEH C18 (1.0×50 mm 1.7 μm particle size), BEH C18 (2.1×50 mm 1.7 μm particle size), and the recent CSH C18 (2.1×50 mm 1.7 μm particle size), were evaluated for their separation of the neuropeptides by liquid chromatography with a binary gradient between water (0.1 %
mean area of plasma samples spiked post-extraction rather than over pure standard solution to limit the contribution of the endogenous background. Matrix effect and extraction recovery parameters for plasma and brain extracts have been previously compared for GnRHs and similar results were observed [36]. Therefore, extensive method validation experiments were run only for the plasma matrix and similarity for the brain extract was assumed. The extraction recovery parameter was estimated by the ratio of the concentrations measured in extracted spiked plasma to extracted plasma spiked post-extraction after endogenous background subtraction. Matrix effect parameter (ME) was evaluated through the ratio of peak area post-extraction after blank subtraction to the peak area in standard solution as described by Matuszewki et al. [37]. The accuracy and precision were validated in five replicates of three concentrations over three consecutive days. Intraday parameters were estimated by the comparison of five replicates analyzed in the same day while interday parameters were based on the data acquired for five replicates over the three validation days. Precision was determined by the coefficient of variation and accuracy by the ratio of the measured concentration to that of spiked standard. All values are expressed with standard deviation range.
Results and discussion Sample preparation and LC separation
110
110
100
100
Relative intensity (%)
Fig. 4 Stability of GnRH and GnIH and related RFamide peptides and internal standards in plasma extract spiked at the concentration of 5 ng/mL for five replicates
Relative intensity (%)
Sample preparation was developed to satisfy the quantitative analyses of all lamprey GnRHs and GnIH and related
90
80 Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2 Lamprey PXRFa-1a Lamprey PXRFa-1b
70
60
90
80 Lamprey GnRH-I Lamprey GnRH-II Lamprey GnRH-III Grass Puffer LP Hagfish RP1 LHRH
70
60
50
50 0
1
2
3 Time (hour)
4
5
0
1
2
3
4
Time (hour)
Relative standard error (%) Time (hour) LFa1a LFa2 LRP1 LFa1b LRP2 GLP LFa HRP1 LHRH GnRH I GnRH II GnRH III 0 2.6 2.5 2.5 2.4 5.2 4.6 4.8 5.1 3.8 4.8 4.5 5.7 1 4.7 1.9 2.7 2.9 6.3 7.0 6.7 6.2 5.9 8.0 7.1 5.3 2 2.3 1.5 2.9 2.8 4.8 5.4 3.2 4.4 4.7 4.5 4.4 4.5 3 2.8 2.1 1.5 1.8 2.1 1.2 2.2 2.2 1.7 2.1 1.5 3.0 4 1.9 1.7 1.0 2.1 1.3 1.3 2.4 1.3 2.0 1.8 1.1 5.1 5 2.1 1.5 1.7 2.4 1.6 1.6 2.0 0.8 1.5 1.4 0.9 1.8
5
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Table 2 Summary of the MSMS parameters for the three GnIHs and three related RFamide peptides, three GnRHs and three internal standards (ISs)
GnIHs and related peptides
GnRHs
ISs
CV (V)
CE (V)
Parent m/z (Da)
Parent charge state
Daughter m/z (Da)
Daughter attribution
Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2
40 40 40 40
20 10 10 25
732.5 436.4 394.0 667.4
[M+3H]3+ [M+4H]4+ [M+3H]3+ [M+2H]2+
897.5 509.4 481.4 546.8
[Y5 +2H]2+ [Y11 +3H]3+ [Y7 +2H]2+ [Y4 +H]+
Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III LHRH Grass puffer LP Hagfish RP1
40 40 40 40 40 40 40 40
15 15 20 15 23 20 25 20
495.5 478.8 613.9 610.9 630.1 591.8 731.0 533.9
[M+3H]4+ [M+5H]5+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+3H]3+ [M+2H]2+
579.0 562.6 741.7 1048.9 773.5 748.0 888.8 848.6
[B12 +2H]2+ [Y20 +4H]4+ [B6 +H]+ [B8 +H]+ [B6 +H]+ [Y7 +H]+ [Y7 +H]+ [Y7 +H]+
formic acid) and acetonitrile. CSH phase is recommended for peptide analyses due to its positively charged Si-stationary phase showing better performances than the other two columns regarding to the peak shape and sensitivity. As mentioned by Chambers et al. [34, 35] each sample set was preceded by nine injections of rat-precipitated plasma to enhance peak shape and sensitivity. Liquid chromatography separation was achieved within 8.5 min, repeatability of retention times was insured by the comparison of spiked extracted plasma, and standard solution displayed in Fig. 1 (Table 2). MSMS parameters MSMS analysis is a challenging step during LC-MS/MS determination of peptides in biological samples. To address this issue, ionization and collision-induced dissociation (CID) condition were optimized for GnRH and GnIH and related RFamide peptide analyses with a Waters TQ-S triple quadrupole mass spectrometer. Instrument resolution was decreased to monitor multiple charged compounds. MRM parameters
were manually optimized with 10 μL injections of 1 μg/mL solutions. For each peptide, collision energy (CE) was optimized for the highest intensities of charge states. Although some studies have resulted in the successful use of low m/z product ions with good sensitivity [29–31], the selection of ions with higher m/z, especially in the case of multiple charged macro molecules such as peptides, tends to lead to better selectivity [32]. A single transition was selected for each peptide with respect to sensitivity and specificity. In fact, two types of transitions were investigated and split into two categories; i) the smallest m/z daughter ions (200 Da) produced under lower CID energies. Transitions exploiting low energies are summarized in Table 1 while transition exploiting higher energies were used as follows: parent m/z > daughter m/z (collision energy/ V); Lamprey PQRFa 732.5 >129.3 (50), Lamprey PQRFa-2 436.4>109.7 (40), Lamprey PQRFa-RP1394.0>119.9 (40), Lamprey PQRFa-RP2 667.4>119.8 (50), Lamprey PXRFa1a 495.5>119.8 (30), Lamprey PXRFa-1b 478.8>119.9 (30), Lamprey GnRH I 614.0>173.0 (45), Lamprey GnRH II
Table 3 General performances of the determination of GnRHs and GnIHs and related RFamide peptides by LC-MSMS in biological matrix
GnIHs and related peptides
GnRHs
Linear range
R2
LOD (ng/mL)
LOQ (ng/mL)
Retention time (min)
Lamprey PQRFa Lamprey PQRFa-2
0.1–50 0.025–50
0.9979 0.9983
0.1 0.05
0.5 0.1
5.41 2.48
Lamprey PQRFa-RP1 Lamprey PQRFa-RP2 Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III
0.01–50 0.075–50 0.025–50 0.05–50 0.01–50 0.01–50 0.250–50
0.9989 0.9978 0.9997 0.9978 0.9979 0.9985 0.9987
0.025 0.1 0.1 0.075 0.1 0.025 0.25
0.1 0.25 0.25 0.25 0.25 0.1 0.75
3.42 4.45 2.70 2.56 3.29 4.60 2.54
Simultaneous determination of GnIH and GnRH
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Table 4 Matrix effect and recovery parameters for GnIH and related RFamide, GnRH and internal standard peptides
GnIHs and related peptides
GnRHs
ISs
Matrix effect (%) [mean±SD]
Recovery (%) [mean±SD]
Selected IS(s)
Lamprey PQRFa Lamprey PQRFa-2 Lamprey PQRFa-RP1 Lamprey PQRFa-RP2
98.8±4.0 91.4±6.3 84.7±6.0 98.3±4.0
81.7±4.9 79.6±3.9 64.1±4.9 79.7±4.9
GLP GLP HRP1 GLP
Lamprey PXRFa-1a Lamprey PXRFa-1b Lamprey GnRH I Lamprey GnRH II Lamprey GnRH III LHRH Grass Puffer LP Hagfish RP1
75.5±2.6 70.3±1.3 94.9±3.4 91.3±5.1 100.3±11.2 99.0±3.6 94.8±4.8 76.1±4.3
90.1±6.7 104.5±7.3 88.2±4.3 72.9±2.3 74.3±5.7 80.7±3.3 85.5±6.1 68.3±12.7
GLP and HRP1 GLP and HRP1 LHRH LHRH LHRH GnRH’s IS GnIH’s IS GnIH’s IS
Table 5 Intra- and interday accuracy and precision of the determination of GnIHs, related RFamides, and GnRHs by LC-MSMS in biological matrix Intraday (n=5) Added (ng mL−1) GnIHs and related peptides
Lamprey PQRFa
Lamprey PQRFa-2
Lamprey PQRFa-RP1
Lamprey PQRFa-RP2
Lamprey PXRFa-1a
GnRHs
Lamprey PXRFa-1b
Lamprey GnRH I
Lamprey GnRH II Lamprey GnRH III
a
Accuracy=100[determined concentration/added]
c
Coefficient of variation
[mean±SD]a (ng mL−1)
Accuracyb (%)
Precisionc (%)
[mean±SD]a (ng mL−1)
Accuracyb (%)
Precisionc (%)
0.50
0.44±0.06
88.0
13.6
0.48±0.09
96.7
18.4
5.00
4.85±0.40
96.7
8.3
5.34±0.61
106.8
12.3
50.00
56.14±4.83
112.3
8.6
53.08±5.84
106.2
11.7
0.50
0.56±0.09
111.2
16.7
0.53±0.08
106.4
16.4
5.00
4.74±0.54
94.9
11.4
4.81±0.56
96.1
11.2
50.00
53.47±7.34
106.9
13.7
51.71±6.84
103.4
13.7
0.50
0.54±0.05
108.0
9.4
0.51±0.07
101.2
13.3
5.00
4.18±0.65
83.6
15.6
4.28±0.58
85.6
11.5
50.00
43.51±4.96
87.0
11.4
43.6±5.03
87.3
10.1
0.50
0.46±0.07
91.6
15.7
0.45±0.07
90.4
13.5
5.00
4.65±0.72
93.0
15.5
4.58±0.61
91.7
12.1
50.00
46.38±5.41
92.8
11.7
47.15±5.79
94.3
11.6
0.50
0.58±0.09
116.8
16.2
0.57±0.08
113.2
16.6
5.00
5.08±0.56
101.6
11.1
5.31±0.56
106.2
11.2
50.00
54.79±8.20
109.6
15.0
53.24±6.99
106.5
14.0
0.50
0.51±0.08
102.8
15.2
0.51±0.08
101.5
15.6
5.00
4.73±0.57
94.5
12.1
5.11±0.66
102.2
13.2
50.00
55.65±6.60
111.3
11.9
53.66±6.10
107.3
12.2
0.50
0.47±0.05
94.8
11.4
0.47±0.06
93.2
11.3
5.00
4.10±0.49
82.0
11.9
4.59±0.62
91.8
12.4
50.00
40.97±1.31
81.9
3.2
42.95±3.68
85.9
7.4
0.50
0.41±0.07
81.2
16.8
0.41±0.06
82.9
12.6
5.00
4.57±0.72
91.4
15.8
4.49±0.52
89.7
10.4
50.00
41.80±3.92
83.6
9.4
41.94±3.95
83.9
7.9
5.00
4.02±0.46
80.4
11.4
4.09±0.46
82.0
9.1
50.00
43.58±5.88
87.2
13.5
43.7±3.68
87.4
7.4
Average determined concentration±standard deviation
b
Interday (n=15)
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611.0>173.0 (35), and Lamprey GnRH III 420.0>110.0 (35). The performances were evaluated on five replicates made of a plasma extract spiked with 1 ng/mL of neuropeptides. Figure 2 highlights the approach for the selection of the MRM transition with the example of Lamprey PXRFa-1b. First of all, the full scan spectrum showed the signal dilution into several charge states; secondly, the two different types of collision-induced dissociations. The fragment at 119.9 Da obtained under collision energy of 30 V showed the best sensitivity. On the other hand, the fragment at 562.6 Da obtained under lower collision energy (15 V) was more specific to Lamprey PXRFa-1b. While sensitivity is a key parameter for quantitative analyses, determination methods need to be specific to minimize the noise amplitude. With respect to the quantitative analysis based on MSMS detection, a primary concern remains to be the signal-to-noise ratio (S/N). Therefore, S/N was measured for two types of transitions in plasma spiked with 1 ng/mL of neuropeptides. Experiments were carried out in plasma to allow for the best evaluation of the endogenous background on intensities and S/N. Intensities and S/N were normalized to the higher value for each peptide. As shown in Fig. 3, the best S/N was obtained for the transition at 478.8>562.6. Despite a sensitivity that was twice of the other transition, the transition at 478.8>119.9 showed a S/N five times lower. A similar trend was observed for other peptides (Fig. 3). Six of the nine investigated neuropeptides showed higher intensities for the transition using daughter ions with m/z under 200 Da, showing better sensitivities. Figure 3B shows normalized S/N for all nine peptides with two types of transitions. Contrary to the intensity, all nine peptides displayed higher S/N for the MSMS transition exploiting m/z greater than 200 Da. Opposite to the signal broadening on low-resolution instruments, the MSMS
General performances Limit of determination (LOD) and limit of quantification (LOQ) are defined as S/Ns of 3 and 10, respectively. LOD and LOQ were first evaluated in standard solution and then determined in spiked extracted plasma, and subsequently summarized in Table 2. Relative intensity to the selected internal standard(s) was plotted against the concentrations to build calibration curves from 0.01 to 50 ng/mL. Linearity was validated by the correlation coefficient of linear regression (R2) ranging from 0.9978 to 0.9997 while LOQ in plasma samples were systematically under 1 ng/mL, allowing for the detection of the targeted neuropeptides in brain and plasma matrices of sea lamprey (Table 3). Matrix effect and extraction recovery As expected for LC-MSMS analysis of molecules in biological samples, matrix effects were observed. Matrix effects were evaluated through comparison of peptide solutions made in standard solution and extracted plasma spiked postextraction at the final concentration of 5 ng/mL. Extraction recovery parameters were calculated for each analyte and internal standard based on the ratio of matrix spiked before and after extraction. For GnRHs, LHRH has already been
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Fig. 5 Average concentrations of neuropeptides in plasma (A) and brain tissues (B)
analysis of multiple-charged molecules allows for the selection of daughter ions with larger m/z than the parent ion m/z. Except for lamprey PQRFa-RP2, the selected transitions exploiting this last attribute were summarized in Table 1. The selection of specific MSMS transitions combined with liquid chromatography separation allows for the determination of the targeted peptides without interferences.
Simultaneous determination of GnIH and GnRH
demonstrated as a suitable internal standard. For GnIHs, Grass puffer LP, and Hagfish RP1 were evaluated by comparing matrix effects and extraction recovery parameters. Matrix effects and extraction recoveries determined for lamprey PQRFa, PQRFa-2, and PQRFa-RP2 were similar to those of grass puffer LP while the value determined for lamprey PQRFa-RP1 suggests hagfish RP1 as a more suitable internal standard (Table 1). Lamprey PXRFa-1a and PXRFa-1b showed dichotomous behaviors. Indeed, matrix effect values were similar to that of hagfish RP1 while extraction recovery values were closer to that of grass puffer LP. This justifies the usage of both hagfish RP1 and grass puffer LP as internal standards for lamprey PXRFa-1a and PXRFa-1b (Table 4).
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GnRH III concentration was measured at 1.02±0.50 ng/mL and 12.38±3.19 ng/mL in male and female plasma extracts, respectively. Lamprey PQRFa RP2 and PXRFa 1b were respectively measured at 1.77±0.18 ng/mL and 0.86±0.15 ng/ mL in the male brain and 2.79±0.77 ng/mg and 1.55±0.36 ng/ mL in the female brain. The significant difference between plasma and brain matrices and/or prespermiating male and preovulatory female observed for seven of the nine targeted neuropeptides confirmed the performance of the method and its applicability to biological study as routine analysis.
Conclusion Accuracy and precision Precision and accuracy were evaluated on the same day through analyses of five replicates of three concentrations. The validation run was repeated over three consecutive days. Intraday accuracy ranged from 80.4 to 116.8 % while interday accuracy ranged from 82.0 to 113.2 %, as shown in Table 3. Precision ranged from 3.2 to 16.8 % from intraday analyses and from 7.4 to 18.4 % from interday analyses (Table 5). As required by the FDA guidelines, precision was less than 20 % for the lowest validation concentration (LQC) while the two other concentrations (MQC and HQC) were above 15 %. Moreover, accuracy values do not exceed the limit of ±20 % of the expected value and fulfilled the requirement for biological sample analysis. Method reproducibility and automation is also dependent upon retention time repeatability which were evaluated for each peptide through five replicates at 5 ng/mL over three consecutive days (n=15). The inter day reproducibility of retention time was validated by the standard deviations ranging from 0.01 to 0.08 min with precision ranging from 0.52 to 2.35 % (coefficient of variation) (Table 5). Application The method was used to analyze GnRHs and GnIHs and related RFamide peptides in lamprey plasma and brain tissues. With respect to the expression of neuropeptides in brain, the determined concentrations were expressed relative to wet tissue mass, which ranged from 41.3 to 90.0 mg. Animals were grouped by sex and maturation corresponding to prespermiating males and preovulatory females. Large variations were expected as supported by interindividual variation and peptide stability. Five GnIHs and related RFamide peptides and two GnRHs were determined in biological matrix. Differences were observed between plasma and brain tissues (Fig. 5). Highest concentrations were measured for GnRH III in plasma while lamprey PQRFa-RP2 and PXRFa-1b were the most abundant in the brain. Lamprey
The full analytical approach for the determination of six GnIHs and related RFamide peptides and three GnRHs have been optimized, validated, and applied to biological samples. The study resulted in a rapid, precise, selective, and sensitive method for the determination of nine neuropeptides that play key roles in the gonadotropin regulation. This is the first study using SPE/LC-MSMS to quantify GnIH and related RFamide peptides. In addition, simultaneous determination of both GnIH and GnRH had never been reported. Matrix effect, recovery, and accuracy and precision parameters were evaluated while optimizing the selectivity and sensitivity combination in biological samples. This method was applied in analysis of sea lamprey brain tissue. Positive results are also expected on plasma matrix since they have shown higher levels of GnRHs [36, 39]. Analytical performances described here suggest that the method could be employed for routine analysis of biological samples. With slight modification, this methodology could be adapted to determine neuropeptides in other vertebrate species including humans. Acknowledgments The authors thank Professor Daniel Jones and Lijun Chen of the Michigan State University MS Facility for helpful advice. This study was funded by a grant from the Great Lakes Fishery Commission.
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