Metabolism of Growth Hormone Releasing Peptides - Analytical

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The metabolism of growth hormone releasing peptides (GHRPs) Andreas Thomas, Philippe Delahaut, Oliver Krug, Wilhelm Schänzer, and Mario Thevis Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Oct 2012 Downloaded from http://pubs.acs.org on October 29, 2012

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Analytical Chemistry

The metabolism of growth hormone releasing peptides (GHRPs) Andreas Thomas1*, Philippe Delahaut2, Oliver Krug1, Wilhelm Schänzer1, Mario Thevis1

1

Center for Preventive Doping Research / Institute of Biochemistry, German Sport University

Cologne, Germany 2

CER Groupe - Département Santé, Rue du Point du Jour, 8, Marloie, Belgium

*Correspondence Andreas Thomas, PhD Center for Preventive Doping Research / Institute of Biochemistry German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne Germany Tel.: 0221-49827072 Fax: 0221-49827071 [email protected]

ACS Paragon Plus Environment

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Abstract New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising 4-7 amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, Hexarelin, Alexamorelin and Ipamorelin. With the exemption of GHRP2, the entity of these peptides represents non-approved pharmaceuticals; however, via internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (p.o. and i.v. administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation exchange solid-phase extraction and analysed by UHPLC-separation followed by high resolution / high accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyser for the first metabolite screening and the speed of a quadrupole / time of flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high accuracy product ion scan experiments. Two deuterium-labelled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimise the extraction and analysis procedure. Overall, 28 metabolites (at least 3 for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase- or endopeptidase activity.


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Introduction Recently, a class of small peptides with known up-regulating effect on endogenous growthhormone (hGH) production and secretion has received considerable attention of doping control authorities.

1-5

Due to their biological effects, these compounds are summarised as GH-releasing

peptides (GHRP) and their analysis in a doping control setting is not accomplished by means of established screening assays for GH e.g. based on the differential isoform test.6 Findings of such GHRPs in nutritional supplements and the availability by internet/black market providers demonstrate the necessity to develop test methods allowing for the efficient detection of these substances and/or their degradation products.5, 7 Due to their low molecular mass ( 20 000 FWHM, particularly when comparably large amounts of the intact peptide are present. Thus, LC-fractionation was performed in order to separate the parent drug from its metabolite enabling the identification of the latter by accurate mass product ion scan experiments. Alternatively to a deamidation process, the observed increment of 1 Da is explainable by the transfer of a hydroxyl group to the ε-amino function of


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the C-terminal lysine residue present in case of GHRP-1, -2, -6, Hexarelin, Alexamorelin and Ipamorelin.3 In order to verify or falsify the proposed deamidation process, the ratio of the ionisation response in positive and negative ionisation mode was evaluated. Here, the Q Exactive benchtop Orbitrap mass spectrometer operated in scan-to-scan polarity switch mode was used. Figure 3 shows exemplarily the chromatograms for the analysis of two combined LC-fractions of the in vitro experiments of GHRP-1 containing the two metabolites M1 and M5. If M5 is the deamidation product of M1, this metabolite should be more acidic and yield a better ionisation response in negative mode due to a carboxylic acid at the C-terminus. This phenomenon is indeed found as demonstrated in Figure 3 by an approximately tenfold increased ionisation efficiency for M5 (R-/R+= 0.177 for M5 and 0.020 for M1) corroborating the hypothesis of deamidation rather than deamination (at ε-position of Lys) at the C-terminus accordingly. To our best knowledge, such a simple experiment to distinguish between an amidated or carboxylic C-terminus in a peptide was not described before. Thus, the proof-of-principle was additionally tested for the two model peptides (TrpAlaTrpPheLys and TrpAlaTrpPheLys-NH2), yielding confirmatory results with a considerably decreased ionisation efficiency in negative mode for the amidated model peptide (data not shown). Despite the fact that this approach does not allow to estimate or calculate absolute gas phase acidity/basicity of peptidic ions, relative ionisation efficiencies (R/R+) of different peptides with mass spectrometry were obtained and support structural interpretation. 21, 22 Finally, one of the responsible enzymes for the metabolism was investigated with in vitro experiments using recombinant amidase. Figure 4 illustrates the effectiveness of this enzyme for the deamidation of GHRP-2, Hexarelin and Ipamorelin. Especially for GHRP-2 and Hexarelin the turnover was found to be nearly complete (~ 95%) and, thus, this approach could potentially serve as an interesting tool to generate reference material for the main metabolites without chemical synthesis. Additional degradation processes were not observed in these experiments when monitoring the respective ion traces. For Ipamorelin the deamidation process under the described conditions is less effective, which is obvious with regard to the relative intensities of the diagnostic signals for Ipamorelin and its deamidated metabolite in Figure 4f).


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GHRP-2 metabolism In the investigated group of compounds, GHRP-2 represents an extraordinary candidate as it is the only clinically approved pharmaceutical agent (Kaken 100). Comprising three modified amino acid residues (and an amidated C-terminus), its bioavailability is described after oral as well as i.v. application. Moreover, a potential masking effect concerning doping control detection assays for hGH employing the accepted GH assay were described.13 Due to the established metabolism in humans, this peptide was not included in the present in vivo studies. Former studies showed the renal elimination of minor amounts of the parent drug as well as one major metabolite after intravenous administration.3, 4, 13 Two additional metabolites were described but their abundance in urine was considered negligible.3 In the in vitro experiments of the present study using human serum, another two metabolites were identified as illustrated in Figure 4. In accordance to the findings for the other GHRPs, the C-terminal deamidation was confirmed. The evidence of this structure proposal (deamidation of the C-terminus instead of deamination of the ε-amino function of the lysine residue) was given by HRMS product ion scan experiments of the [M-H]- ion at m/z 817.40 in negative ionisation mode from the LC-fractionated in vitro sample (data not shown). By LC-fractionation it was possible to separate the excess of non-metabolised GHRP-2 from its deamidated metabolite (M1) to yield sufficient mass spectrometric information for proper identification. Interestingly, an amidated metabolite (D-Ala)(β-Nal)Ala-NH2 (M3) was also found in minor extent in the incubated serum samples. Its occurrence is not explainable by a linear exopeptidic degradation process as described for the other analogues before. This unusual finding strongly suggests that the metabolism of GHRP-2 is due to endopeptidase activity in addition to exopeptidases as selected endopeptidases are described to induce amidation during the peptide bond cleavage process.

15-18, 23

Both new metabolites (together with the former established M2)

were also confirmed to be present in human excretion study urine samples (see Figure 5) by analysis with sensitive product ion scan experiments on the Q Exactive mass spectrometer. Here it is obvious, that the amidated metabolite M3 is present only in trace amounts, but in addition to the well established M2, the deamidated substrate (M1) is excreted in considerable extent (estimated roughly by the peak areas in the extracted product ion chromatograms). Intact GHRP-2 was found in this sample only in trace concentrations.


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Conclusion The investigation of metabolic processes for peptide hormones represents a new challenge in doping controls and analytical chemistry. The combination of animal in vivo and human in vitro approaches together with hyphenated mass spectrometric detection systems enables an efficient study design in order to identify and characterise potential metabolites. Within this study important qualitative data are provided for doping control laboratories to implement a facile LCMS/MS-based procedure for these prohibited compounds by means of commonly and routinely employed instrumental equipment (triple quadrupole mass spectrometers or equivalent). Although derived from animal in vivo studies, the model appears valid in a qualitative manner since human in vitro incubations corroborated the likely presence of the surrogate in vivo generated compounds in human urine. Furthermore, with the same procedure it is also possible to determine additional prohibited peptides in urine (e.g. desmopressin) and, thus, the method provides the option to implement a screening assay for a variety of different compounds instead of methods limited to individual compounds only. 24 Here it would be interesting to develop a sophisticated purification procedure to detect the target analytes also in blood. This is of particular interest as doping control tests for hGH are conducted with serum. In case of atypically high amounts of endogenous hGH, the same sample can/should be analysed for the presence of growth hormone releasing peptides.

Acknowledgements The study was carried out with support of the Manfred Donike Institute for Doping Analysis, Cologne, Germany, the Federal Ministry of the Interior of the Federal Republic of Germany, and the World Anti-Doping Agency (WADA, Grant #10B10MT).

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.


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(5)

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(6)

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(7)

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(8)

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(9)

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(10)

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(11)

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(12)

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(14)

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(15)

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(17)

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(18)

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(19)

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(20)

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(21)

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(22)

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(23)

Kim, K. H.; Seong, B. L. Biotechnol. Bioprocess. Eng. 2001, 6, 244-251.

(24)

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XIC of +TOF Product (608.3): Exp 2, 334.131 to 334.181 Da 11.47 WAWF-NH2 8000

11.66

DWAWDF-NH2

GHRP-4 Intensity, cps 9.8

10.2

10.6

11.0

11.4 11.8 Time, min

+TOF Product (608.3): WAWF-NH2

a1 100%

258.1245

159.0922

b2

12.2

12.6

13.0

13.4

+TOF Product (608.3): DWAWDF-NH2 444.2037

334.1567 y2-NH3

b3 1100%

b2

a1

y2-NH3

159.0920

334.1561

Rel. Int. (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


258.1252

444.2053

306.1607

351.1833 306.1614

591.2734

230.1291

260

591.2753

230.1290

351.1812

180

b3

340 420 m/z, Da

500

580

180

260

340 420 m/z, Da

500

580

Figure 1: Chromatographic separation of D/L-amino acid containing GHRP-4 and its natural analogue by UHPLC.


15


TOF Product (612.2): Exp 5, 334.128 to 334.178 Da

TOF Product (361.1): Exp 3, 170.072 to 170.122 Da 9.85 5314

11.34

7982

ISTD 1

ISTD 2

8.5

9.5

10.5

11.5

12.5

7.5

8.5

9.5

10.5

TOF Product (368.7): Exp 6, 159.065 to 159.115 Da 9.93 8357 Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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DWAWDFK-NH 2 (M3)

7.5

8.5

9.5

10.41

10.5

DWAWDFK (M2)

11.5

12.5

13.5

12.5

13.5

TOF Product (437.2): Exp 7, 324.122 to 324.172 Da 9.06 1.5e4

H DWAWDFK-NH2 (GHRP-6) 7.5

8.5

9.69 9.5

HDWAWDFK (M1)

10.5

11.5

TOF Product (609.3): Exp 2, 335.111 to 335.161 Da

335.1401

100%

11.55 1.8e4

a1

DWAWDF

159.0916 258.1238

(M4) 8.5

9.5

10.5

11.5 Time, min

y2-NH 2

y2 352.1664

609.37

b3 444.2049

150

7.5

b2 248.1685

12.5

250

350

450

550

13.5

Figure 2: Extracted ion chromatograms of product ion scans aiming at GHRP-6 metabolites in a rat urine sample collected 9 hours after intravenous administration of GHRP-6 (G5-IV-9-R1-9h). Abundant signals are observed for ISTD 1+2, GHRP-6 and the metabolites of GHRP-6 M1-M4. Small inset give an example for the corresponding product ion spectra.


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100

m/z= 883.39-883.442 - p ESI Full ms

RT: 6.54 100

NL: 7.95E4

883.4282

R-/R+=0.177 884.4309 885.4321

100

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RT: 6.53

m/z= 443.22-443.23 + p ESI Full ms

NL: 4.50E5

HDA(nA)AWDFK M5

100

100

RT: 5.94

NL: 8.78E4

m/z= 882.43-882.46 - p ESI Full ms

R-/R+=0.020

RT: 5.93

NL: 4.41E6

m/z= 442.73-442.74 + p ESI Full ms

HDA(nA)AWDFK-NH2 M1

4.0

5.0

6.0 Time (min)

7.0

Figure 3: Comparison of ionisation response in positive and negative ionisation mode (R-/R+), calculated by the normalised largest (NL) peak heights for the metabolites M1 at 5.9 min and M5 at 6.5 min of GHRP-1. Positive ions (monoisotopic mass) are extracted from the full scan chromatograms as [M+2H]2+ and negative ions as [M-H]-. Small inset give an example for the corresponding high resolution mass spectrum of M5 in negative full scan mode yielding mass accuracies < 3 ppm.


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Incubation without amidase

Incubation with amidase (EC 3.5.1.4)

10.72 1.8e6

a)

2.4e6

GHRP-2 DA(nA)AWDFK-NH 2

11.02 b)

Intensity, cps

Intensity, cps

GHRP-2 M1 DA(nA)AWDFK GHRP-2 DA(nA)AWDFK-NH 2

10.72 9.4 5.2e5

10.2

11.0

9.6

11.8

10.4

11.2

8.99

c)

3.2e5

12.0

9.70

12.8

Hexarelin M1 HD(Mrp)AWDFK

d)

Hexarelin HD(Mrp)AWDFK-NH2 Intensity, cps

Intensity, cps

Hexarelin HD(Mrp)AWDFK-NH2

8.99 7.0

8.0

9.0

10.0

1.8e5

7.0

11.0

7.90

6.4e4

e)

Ipamorelin AibHD(nA)DFK-NH2

6.0

7.0

Intensity, cps

Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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8.0 Time, min

9.0

10.0

8.0

9.0

10.0

11.0

12.0

8.48 f)

Ipamorelin AibHD(nA)DFK-NH2

6.0

7.0

7.89

8.0 Time, min

Ipamorelin M3 AibHD(nA)DFK

9.0

10.0

11.0

Figure 4: Product ion chromatograms of the in vitro metabolism samples using recombinant amidase (EC 3.5.1.4) for the doubly charged precursors [M+2H]2+of a+b) GHRP-2 (TOF product of m/z 410), c+d) Hexarelin (TOF product of m/z 444) and e+f) Ipamorelin (TOF product of m/z 357). After incubation for 36 hours at 30 °C considerable amounts of the peptides were deamidated yielding the respective metabolites.


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RT: 11.86

100 Full ms2 358.1 m/z= 170.09

NL: 2.1E6 DA(nA)A

M2 100 Full ms2 357.1 m/z= 170.09

RT: 11.16 100

NL: 1.7E4

170.0957 241.1327

M3

120.0807 198.0898 160

100

Rel. Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

240

306.5742 357.1786 320

RT: 11.17

NL: 1.2E4

m/z= 241.13 DA(nA)A-NH2

M3 100

Full ms2 410.2 m/z= 294.18

170.0960

RT: 13.23

NL: 1.8E5

RT: 13.23

NL: 7.1E5

M1 241.1327294.1804 159.0914 198.0907 120.0807 306.1582 200

410.3043 380

100 m/z= 170.09 DA(nA)AWDFK

M1 11.0

11.4

11.8

12.2 Time (min)

12.6

13.0

13.4

Figure 5: Extracted ion chromatograms of product ion scans aiming ion chromatograms of GHRP-2 metabolites (M1 at m/z 410.2, M2 at m/z 358.2 and M3 at m/z 357.2) and product ion spectra of M1 and M3 (small insets) from an excretion study urine sample (5 h after 10 mg p.o.).


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

name

GHRP-2 GHRP-1 GHRP-6 GHRP-5 GHRP-4 Alexamorelin Hexarelin Ipamorelin GHRP-2 metabolite ISTD1 ISTD2

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amino acid sequence

monoisotopic mass elemental

(D-Ala)-(D-β-Nal)-Ala-Trp-(D-Phe)-Lys-NH2 Ala-His-(D-β-Nal)-Ala-Trp-(D-Phe)-Lys-NH2 His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2 Tyr-(D-Trp)-Ala-Trp-(D-Phe)-NH2 (D-Trp)-Ala-Trp-(D-Phe)-NH2 Ala-His-(D-Mrp)-Ala-Trp-(D-Phe)-Lys-NH2 His-(D-Mrp)-Ala-Trp-(D-Phe)-Lys-NH2 Aib-His-(D-β-Nal)-(D-Phe)-Lys-NH2 (D-Ala)-(D-β-Nal)-Ala (D-[2]H3-Ala)-(D-β-Nal)-Ala (D-Trp)- [2]H4-Ala-Trp-(D-Phe)-NH2

[Da] 817.427 954.486 872.444 770.354 607.292 957.497 886.460 711.385 357.168 360.187 611.315

composition C45H55N9O6 C51H62N12O7 C46H56N12O6 C43H46N8O6 C34H37N7O4 C50H63O7N13 C47H58N12O6 C38H49N9O5 C19H23N3O4 C19H20[2]H3N3O4 C34H33[2]H4N7O4

dominant charge state (ESI) 2+ 2+ 2+ 1+ 1+ 2+ 2+ 1+/2+ 1+ 1+ 1+

Table 1: Growth hormone releasing peptides, metabolite for GHRP-2 and the used ISTDs with their amino acid sequence, elemental composition, monoisotopic masses and dominant charge state. (Non-standard abbreviations: Nal=naphtylalanine, Mrp=2methyltryptophane, Aib=aminoisobutyric acid


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For TOC only:

LC-HRMS


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Metabolism of Growth Hormone Releasing Peptides - Analytical

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